pLentiRNACRISPR_005 - hU6-DR_BsmBI-EFS-RfxCas13d-NLS-2A-Pur

LOCUS        pLentiRNACRISPR_005 - hU6-DR_BsmBI-EFS-RfxCas13d-NLS-2A-Puro-WPRE               11734 bp ds-DNA     circular SYN 09-DEC-2021
DEFINITION  Expresses RfxCas13d in mammalian cells, localized in the nucleus. 
            For cloning of guide RNAs compatible with RfxCas13d. Contains a 5' 
            direct repeat. Clone using BsmBI. F overhang aaac. R overhang aaaa.
ACCESSION   .
VERSION     .
KEYWORDS    pLentiRNACRISPR_005 - hU6-DR_BsmBI-EFS-RfxCas13d-NLS-2A-Puro-WPRE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11734)
  AUTHORS   Wessels HH, Mendez-Mancilla A, Guo X, Legut M, Daniloski Z, Sanjana 
            NE
  TITLE     Massively parallel Cas13 screens reveal principles for guide RNA 
            design.
  JOURNAL   Nat Biotechnol. 2020 Jun;38(6):722-727. doi: 
            10.1038/s41587-020-0456-9. Epub 2020 Mar 16.
  PUBMED    32518401
REFERENCE   2  (bases 1 to 11734)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 9, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..11734
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1443..1676
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1861..1905
                     /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    2203..2320
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2371..2611
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2371..2391
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2542..2561
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2621..2650
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     promoter        2729..2940
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2965..5862
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDS
                     IRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLE
                     KRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTV
                     YTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYII
                     NYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITN
                     ELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRK
                     DMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFV
                     INLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPA
                     GRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAF
                     FKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDE
                     NGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQ
                     KKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRE
                     NAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLE
                     EKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKK
                     AEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVH
                     AYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCV
                     PFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGN"
     CDS             5863..5910
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5920..5976
                     /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             5977..6573
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     6470..6490
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    6589..7177
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6642..6662)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7060..7071)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7249..7482
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(7508..7525)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7514..7738
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7784..8212
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7871..7890)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8081..8102
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8221..8241)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8226..8555
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      8406..8541
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     8468..8487
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        8603..8650
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             8669..9043
                     /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    9173..9294
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9210..9229)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9264..9283
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(9343..9359)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9343..9359)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9356..9378)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9367..9383
                     /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(9391..9421)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9436..9457
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9574..9591)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9745..10333)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9825..9844)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(10504..11364)
                     /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     11127..11146
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11365..11469)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(11544..11563)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2341 cagagatcca gtttggttaa ttaaggtacc gagggcctat ttcccatgat tccttcatat
     2401 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca
     2461 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     2521 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     2581 ttcttggctt tatatatctt gtggaaagga cgaaacaccg aacccctacc aactggtcgg
     2641 ggtttgaaac ggagacggac gtctcttttt tttgaattcg ctagctaggt cttgaaagga
     2701 gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     2761 aagttggggg gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac
     2821 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     2881 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     2941 gaccggttct agagcgctgc caccatgatc gaaaaaaaaa agtccttcgc caagggcatg
     3001 ggcgtgaagt ccacactcgt gtccggctcc aaagtgtaca tgacaacctt cgccgaaggc
     3061 agcgacgcca ggctggaaaa gatcgtggag ggcgacagca tcaggagcgt gaatgagggc
     3121 gaggccttca gcgctgaaat ggccgataaa aacgccggct ataagatcgg caacgccaaa
     3181 ttcagccatc ctaagggcta cgccgtggtg gctaacaacc ctctgtatac aggacccgtc
     3241 cagcaggata tgctcggcct gaaggaaact ctggaaaaga ggtacttcgg cgagagcgct
     3301 gatggcaatg acaatatttg tatccaggtg atccataaca tcctggacat tgaaaaaatc
     3361 ctcgccgaat acattaccaa cgccgcctac gccgtcaaca atatctccgg cctggataag
     3421 gacattattg gattcggcaa gttctccaca gtgtatacct acgacgaatt caaagacccc
     3481 gagcaccata gggccgcttt caacaataac gataagctca tcaacgccat caaggcccag
     3541 tatgacgagt tcgacaactt cctcgataac cccagactcg gctatttcgg ccaggccttt
     3601 ttcagcaagg agggcagaaa ttacatcatc aattacggca acgaatgcta tgacattctg
     3661 gccctcctga gcggactgag gcactgggtg gtccataaca acgaagaaga gtccaggatc
     3721 tccaggacct ggctctacaa cctcgataag aacctcgaca acgaatacat ctccaccctc
     3781 aactacctct acgacaggat caccaatgag ctgaccaact ccttctccaa gaactccgcc
     3841 gccaacgtga actatattgc cgaaactctg ggaatcaacc ctgccgaatt cgccgaacaa
     3901 tatttcagat tcagcattat gaaagagcag aaaaacctcg gattcaatat caccaagctc
     3961 agggaagtga tgctggacag gaaggatatg tccgagatca ggaaaaatca taaggtgttc
     4021 gactccatca ggaccaaggt ctacaccatg atggactttg tgatttatag gtattacatc
     4081 gaagaggatg ccaaggtggc tgccgccaat aagtccctcc ccgataatga gaagtccctg
     4141 agcgagaagg atatctttgt gattaacctg aggggctcct tcaacgacga ccagaaggat
     4201 gccctctact acgatgaagc taatagaatt tggagaaagc tcgaaaatat catgcacaac
     4261 atcaaggaat ttaggggaaa caagacaaga gagtataaga agaaggacgc ccctagactg
     4321 cccagaatcc tgcccgctgg ccgtgatgtt tccgccttca gcaaactcat gtatgccctg
     4381 accatgttcc tggatggcaa ggagatcaac gacctcctga ccaccctgat taataaattc
     4441 gataacatcc agagcttcct gaaggtgatg cctctcatcg gagtcaacgc taagttcgtg
     4501 gaggaatacg cctttttcaa agactccgcc aagatcgccg atgagctgag gctgatcaag
     4561 tccttcgcta gaatgggaga acctattgcc gatgccagga gggccatgta tatcgacgcc
     4621 atccgtattt taggaaccaa cctgtcctat gatgagctca aggccctcgc cgacaccttt
     4681 tccctggacg agaacggaaa caagctcaag aaaggcaagc acggcatgag aaatttcatt
     4741 attaataacg tgatcagcaa taaaaggttc cactacctga tcagatacgg tgatcctgcc
     4801 cacctccatg agatcgccaa aaacgaggcc gtggtgaagt tcgtgctcgg caggatcgct
     4861 gacatccaga aaaaacaggg ccagaacggc aagaaccaga tcgacaggta ctacgaaact
     4921 tgtatcggaa aggataaggg caagagcgtg agcgaaaagg tggacgctct cacaaagatc
     4981 atcaccggaa tgaactacga ccaattcgac aagaaaagga gcgtcattga ggacaccggc
     5041 agggaaaacg ccgagaggga gaagtttaaa aagatcatca gcctgtacct caccgtgatc
     5101 taccacatcc tcaagaatat tgtcaatatc aacgccaggt acgtcatcgg attccattgc
     5161 gtcgagcgtg atgctcaact gtacaaggag aaaggctacg acatcaatct caagaaactg
     5221 gaagagaagg gattcagctc cgtcaccaag ctctgcgctg gcattgatga aactgccccc
     5281 gataagagaa aggacgtgga aaaggagatg gctgaaagag ccaaggagag cattgacagc
     5341 ctcgagagcg ccaaccccaa gctgtatgcc aattacatca aatacagcga cgagaagaaa
     5401 gccgaggagt tcaccaggca gattaacagg gagaaggcca aaaccgccct gaacgcctac
     5461 ctgaggaaca ccaagtggaa tgtgatcatc agggaggacc tcctgagaat tgacaacaag
     5521 acatgtaccc tgttcagaaa caaggccgtc cacctggaag tggccaggta tgtccacgcc
     5581 tatatcaacg acattgccga ggtcaattcc tacttccaac tgtaccatta catcatgcag
     5641 agaattatca tgaatgagag gtacgagaaa agcagcggaa aggtgtccga gtacttcgac
     5701 gctgtgaatg acgagaagaa gtacaacgat aggctcctga aactgctgtg tgtgcctttc
     5761 ggctactgta tccccaggtt taagaacctg agcatcgagg ccctgttcga taggaacgag
     5821 gccgccaagt tcgacaagga gaaaaagaag gtgtccggca ataagcgacc tgccgccaca
     5881 aagaaggctg gacaggctaa gaagaagaaa ggatccggcg caacaaactt ctctctgctg
     5941 aaacaagccg gagatgtcga agagaatcct ggaccgaccg agtacaagcc cacggtgcgc
     6001 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     6061 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     6121 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     6181 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     6241 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     6301 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga
     6361 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     6421 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     6481 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     6541 acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg
     6601 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     6661 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     6721 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     6781 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     6841 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     6901 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     6961 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     7021 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     7081 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     7141 cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt
     7201 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     7261 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     7321 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     7381 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     7441 gatccctcag acccttttag tcagtgtgga aaatctctag cagggcccgt ttaaacccgc
     7501 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     7561 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     7621 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     7681 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     7741 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     7801 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     7861 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     7921 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     7981 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     8041 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     8101 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     8161 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     8221 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     8281 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     8341 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     8401 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     8461 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     8521 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     8581 ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt
     8641 ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct
     8701 caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg
     8761 ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag
     8821 cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct
     8881 ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg
     8941 gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc
     9001 ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt
     9061 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     9121 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     9181 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     9241 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     9301 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     9361 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     9421 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     9481 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     9541 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     9601 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     9661 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     9721 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     9781 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     9841 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     9901 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     9961 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
    10021 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
    10081 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
    10141 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
    10201 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
    10261 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
    10321 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
    10381 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
    10441 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
    10501 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
    10561 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
    10621 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
    10681 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
    10741 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
    10801 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
    10861 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
    10921 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
    10981 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
    11041 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
    11101 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
    11161 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
    11221 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
    11281 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
    11341 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
    11401 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
    11461 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc
    11521 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
    11581 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
    11641 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
    11701 ctgcttcgcg atgtacgggc cagatatacg cgtt
//