LentiSAMPHv2

LOCUS       LentiSAMPHv2               14988 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  All-in-one lentiviral CRISPRa gRNA expression with blasticidin 
            selection marker..
ACCESSION   .
VERSION     .
KEYWORDS    LentiSAMPHv2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14988)
  AUTHORS   Chan W, Gottschalk RA, Yao Y, Pomerantz JL, Germain RN
  TITLE     Efficient Immune Cell Genome Engineering with Enhanced CRISPR 
            Editing Tools.
  JOURNAL   Immunohorizons. 2021 Feb 23;5(2):117-132. doi: 
            10.4049/immunohorizons.2000082.
  PUBMED    33622708
REFERENCE   2  (bases 1 to 14988)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..14988
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(78..97)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        269..648
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        650..848
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     803..823
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             866..1046
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1093..1218
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1711..1944
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2129..2173
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2471..2588
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2638..2878
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2638..2658
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2809..2828
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2924..2942
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2994..3012
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        3099..3310
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             3327..3374
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3375..7475
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7530..7550
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7575..7724
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             7746..7802
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7803..8189
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             8244..8264
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8280..8822
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             8847..9218
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     CDS             9234..9287
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             9288..9686
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    9711..10299
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9764..9784)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10182..10193)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10302..10318)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10303..10319)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10824..11004
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(11030..11047)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11036..11260
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11306..11734
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11393..11412)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11603..11624
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11743..11763)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11748..12077
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11928..12063
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11990..12009
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12125..12172
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12191..12565
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    12695..12816
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12732..12751)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12786..12805
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12865..12881)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12865..12881)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12878..12900)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12889..12905
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(12913..12943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12958..12979
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13096..13113)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13267..13855)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13347..13366)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(14026..14886)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     14649..14668
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14887..3)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
       61 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
      121 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
      181 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
      241 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
      301 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      361 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      421 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      481 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      541 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      601 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      661 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      721 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      781 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      841 ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga
      901 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
      961 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     1021 ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg
     1081 aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc
     1141 gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga
     1201 gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt
     1261 cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg
     1321 gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa
     1381 atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat
     1441 aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa
     1501 gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc
     1561 gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg aattatataa
     1621 atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt
     1681 ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc
     1741 agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt
     1801 gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct
     1861 gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag
     1921 atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac
     1981 cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca
     2041 cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt
     2101 aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa
     2161 atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt
     2221 cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt
     2281 gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag
     2341 gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc
     2401 cattcgatta gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa atggcagtat
     2461 tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag
     2521 tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc
     2581 aaaattttcg ggtttattac agggacagca gagatccagt ttggttaatt agctagcgag
     2641 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
     2701 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
     2761 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
     2821 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
     2881 aacaccggag acgggatacc gtctctgttt tagagctagg ccaacatgag gatcacccat
     2941 gtctgcaggg cctagcaagt taaaataagg ctagtccgtt atcaacttgg ccaacatgag
     3001 gatcacccat gtctgcaggg ccaagtggca ccgagtcggt gctttttttg gatcctaggt
     3061 cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
     3121 agtccccgag aagttggggg gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg
     3181 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
     3241 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
     3301 cagaacacag gcgtacggcc accatgaaaa ggccggcggc cacgaaaaag gccggccagg
     3361 caaaaaagaa aaaggacaag aagtacagca tcggcctggc catcggcacc aactctgtgg
     3421 gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca
     3481 acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg
     3541 aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga
     3601 accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct
     3661 tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc
     3721 ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc
     3781 acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg
     3841 ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg
     3901 acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg
     3961 aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga
     4021 gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc
     4081 tgttcggcaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg
     4141 acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca
     4201 acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt
     4261 ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc
     4321 tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag
     4381 ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga
     4441 acggctacgc cggctacatt gacggcggag ccagccagga agagttctac aagttcatca
     4501 agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg
     4561 acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg
     4621 gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc
     4681 gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca
     4741 ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga
     4801 acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca
     4861 acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt
     4921 acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc
     4981 ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc
     5041 ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact
     5101 ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc
     5161 tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg
     5221 aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga
     5281 aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca
     5341 ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca
     5401 agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga
     5461 tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg
     5521 gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca
     5581 tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg
     5641 agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca
     5701 gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga
     5761 aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc
     5821 agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg
     5881 atgtggacca catcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc
     5941 tgaccagaag cgacaaggcc cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga
     6001 agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt
     6061 tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca
     6121 tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact
     6181 cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca
     6241 ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg
     6301 agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc
     6361 tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg
     6421 acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact
     6481 tcttctacag caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga
     6541 tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg
     6601 gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa
     6661 agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg
     6721 ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc
     6781 ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac
     6841 tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga
     6901 atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca
     6961 agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg
     7021 ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt
     7081 acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc
     7141 tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct
     7201 ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc
     7261 accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca
     7321 atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca
     7381 ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg
     7441 agacacggat cgacctgtct cagctgggag gcgacagcgc tggaggaggt ggaagcggag
     7501 gaggaggaag cggaggagga ggtagcggac ctaagaaaaa gaggaaggtg gcggccgctg
     7561 gatccggacg ggctgacgca ttggacgatt ttgatctgga tatgctggga agtgacgccc
     7621 tcgatgattt tgaccttgac atgcttggtt cggatgccct tgatgacttt gacctcgaca
     7681 tgctcggcag tgacgccctt gatgatttcg acctggacat gctgattaac tgtacagggt
     7741 ccggcgcaac aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac
     7801 cggcttcaaa ctttactcag ttcgtgctcg tggacaatgg tgggacaggg gatgtgacag
     7861 tggctccttc taatttcgct aatggggtgg cagagtggat cagctccaac tcacggagcc
     7921 aggcctacaa ggtgacatgc agcgtcaggc agtctagtgc ccagaagaga aagtatacca
     7981 tcaaggtgga ggtccccaaa gtggctaccc agacagtggg cggagtcgaa ctgcctgtcg
     8041 ccgcttggag gtcctacctg aacatggagc tcactatccc aattttcgct accaattctg
     8101 actgtgaact catcgtgaag gcaatgcagg ggctcctcaa agacggtaat cctatccctt
     8161 ccgccatcgc cgctaactca ggtatctaca gcgctggagg aggtggaagc ggaggaggag
     8221 gaagcggagg aggaggtagc ggacctaaga aaaagaggaa ggtggcggcc gctggatccc
     8281 cttcagggca gatcagcaac caggccctgg ctctggcccc tagctccgct ccagtgctgg
     8341 cccagactat ggtgccctct agtgctatgg tgcctctggc ccagccacct gctccagccc
     8401 ctgtgctgac cccaggacca ccccagtcac tgagcgctcc agtgcccaag tctacacagg
     8461 ccggcgaggg gactctgagt gaagctctgc tgcacctgca gttcgacgct gatgaggacc
     8521 tgggagctct gctggggaac agcaccgatc ccggagtgtt cacagatctg gcctccgtgg
     8581 acaactctga gtttcagcag ctgctgaatc agggcgtgtc catgtctcat agtacagccg
     8641 aaccaatgct gatggagtac cccgaagcca ttacccggct ggtgaccggc agccagcggc
     8701 cccccgaccc cgctccaact cccctgggaa ccagcggcct gcctaatggg ctgtccggag
     8761 atgaagactt ctcaagcatc gctgatatgg actttagtgc cctgctgtca cagatttcct
     8821 ctagtgggca gggaggaggt ggaagcggct tcagcgtgga caccagtgcc ctgctggacc
     8881 tgttcagccc ctcggtgacc gtgcccgaca tgagcctgcc tgaccttgac agcagcctgg
     8941 ccagtatcca agagctcctg tctccccagg agccccccag gcctcccgag gcagagaaca
     9001 gcagcccgga ttcagggaag cagctggtgc actacacagc gcagccgctg ttcctgctgg
     9061 accccggctc cgtggacacc gggagcaacg acctgccggt gctgtttgag ctgggagagg
     9121 gctcctactt ctccgaaggg gacggcttcg ccgaggaccc caccatctcc ctgctgacag
     9181 gctcggagcc tcccaaagcc aaggacccca ctgtctcctg tacaggcagt ggagagggca
     9241 gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg gccaagcctt
     9301 tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca
     9361 tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg
     9421 gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg
     9481 ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg
     9541 gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca
     9601 aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc
     9661 cctctggtta tgtgtgggag ggctaagaat tcgatatcaa gcttatcggt aatcaacctc
     9721 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     9781 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     9841 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     9901 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     9961 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
    10021 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
    10081 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
    10141 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
    10201 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
    10261 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgag
    10321 acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg
    10381 cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt
    10441 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
    10501 gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc
    10561 acacacaagg ctacttccct gattggcaga actacacacc agggccaggg atcagatatc
    10621 cactgacctt tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag
    10681 ccaatgaagg agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc
    10741 cggagagaga agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc
    10801 gagagctgca tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc
    10861 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
    10921 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
    10981 agtcagtgtg gaaaatctct agcagggccc gtttaaaccc gctgatcagc ctcgactgtg
    11041 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
    11101 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
    11161 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
    11221 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
    11281 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
    11341 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
    11401 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
    11461 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
    11521 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
    11581 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
    11641 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
    11701 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
    11761 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
    11821 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
    11881 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
    11941 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
    12001 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
    12061 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc
    12121 acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
    12181 gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc
    12241 cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga
    12301 cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt
    12361 ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga
    12421 gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat
    12481 cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca
    12541 cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt
    12601 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
    12661 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
    12721 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
    12781 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
    12841 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
    12901 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
    12961 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
    13021 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
    13081 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
    13141 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
    13201 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
    13261 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
    13321 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
    13381 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
    13441 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
    13501 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
    13561 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
    13621 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
    13681 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
    13741 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
    13801 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
    13861 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
    13921 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
    13981 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
    14041 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
    14101 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
    14161 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
    14221 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
    14281 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
    14341 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
    14401 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
    14461 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
    14521 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
    14581 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
    14641 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
    14701 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
    14761 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
    14821 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
    14881 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
    14941 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttcc
//