All_in_one_CRISPR/Cas9_LacZ

LOCUS      All_in_one_CRISPR/Cas9_LacZ             10694 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  ""All-in-one"" CRISPR/Cas9 (wt) plasmid for cloning of custom gRNA 
            with blue/white screening.
ACCESSION   .
VERSION     .
KEYWORDS    All_in_one_CRISPR/Cas9_LacZ
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10694)
  AUTHORS   Findlay SD, Vincent KM, Berman JR, Postovit LM
  TITLE     A Digital PCR-Based Method for Efficient and Highly Specific 
            Screening of Genome Edited Cells.
  JOURNAL   PLoS One. 2016 Apr 18;11(4):e0153901. doi: 
            10.1371/journal.pone.0153901. eCollection 2016.
  PUBMED    27089539
REFERENCE   2  (bases 1 to 10694)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..10694
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     144..161
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    278..299
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        314..344
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    352..368
                     /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)."
     primer_bind     376..392
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        412..430
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     412..429
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        442..460
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     591..611
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     762..781
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    825..846
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        861..891
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    899..915
                     /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)."
     primer_bind     923..939
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             935..1213
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEW"
     primer_bind     958..978
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        960..978
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(1026..1044)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1054..1071)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1054..1070)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1063..1085)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     enhancer        1276..1655
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1656..1859
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1810..1830
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        1904..1922
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1936..1945
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1942..6045
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6061..6081
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6082..6108
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(6125..6145)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6130..6459
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6310..6445
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6372..6391
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     regulatory      6517..6526
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             6523..7233
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6668..6686)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6936..6955
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    7249..7822
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(7416..7433)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             7858..8661
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7921..7940)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     8531..8550
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    8793..8914
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8830..8849)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8884..8903
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8963..8979)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8987..9003
                     /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)."
     CDS             9065..9925
                     /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     complement(9283..9302)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10096..10684
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10585..10604
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
       61 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
      121 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
      181 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
      241 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
      301 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
      361 gataacaatt tcacacagga aacagctatg acatgattac gaattgcaac gatttaggtg
      421 acactataga agagaaggaa ttaatacgac tcactatagg gagagagaga gaattaccct
      481 cactaaaggg aggagaagca tgaattcccc agtggaaaga cgcgcaggca aaacgcacca
      541 cgtgacggag cgtgaccgcg cgccgagcgc gcgccaaggt cgggcaggaa gagggcctat
      601 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa
      661 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      721 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      781 taactcgaga cgcggtggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
      841 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
      901 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
      961 attaaccctc actaaaggga acaaaagctg gaggacctcg agggggggcc cggtacccaa
     1021 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     1081 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     1141 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     1201 tggcgaatgg taggacgcgc cctgtagcgg cgcgtctcac gctgcttcgc gatgtacggg
     1261 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     1321 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     1381 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     1441 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     1501 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     1561 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     1621 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     1681 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     1741 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     1801 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
     1861 tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg actcactata
     1921 gggagaccca agcttgccac catggacaag aagtacagca tcggcctgga catcggtacc
     1981 aacagcgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaagttcaag
     2041 gtgctgggca acaccgaccg ccacagcatc aagaagaacc tgatcggcgc cctgctgttc
     2101 gacagcggcg agaccgccga ggccacccgc ctgaagcgca ccgcccgccg ccgctacacc
     2161 cgccgcaaga accgcatctg ctacctgcag gagatcttca gcaacgagat ggccaaggtg
     2221 gacgacagct tcttccaccg cctggaggag agcttcctgg tggaggagga caagaagcac
     2281 gagcgccacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     2341 accatctacc acctgcgcaa gaagctggtg gacagcaccg acaaggccga cctgcgcctg
     2401 atctacctgg ccctggccca catgatcaag ttccgcggcc acttcctgat cgagggcgac
     2461 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     2521 cagctgttcg aggagaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgagc
     2581 gcccgcctga gcaagagccg ccgcctggag aacctgatcg cccagctgcc cggcgagaag
     2641 aagaacggcc tgttcggcaa cctgatcgcc ctgagcctgg gcctgacccc caacttcaag
     2701 agcaacttcg acctggccga ggacgccaag ctgcagctga gcaaggacac ctacgacgac
     2761 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt cctggccgcc
     2821 aagaacctga gcgacgccat cctgctgagc gacatcctgc gcgtgaacac cgagatcacc
     2881 aaggcccccc tgagcgccag catgatcaag cgctacgacg agcaccacca ggacctgacc
     2941 ctgctgaagg ccctggtgcg ccagcagctg cccgagaagt acaaggagat cttcttcgac
     3001 cagagcaaga acggctacgc cggctacatc gacggcggcg ccagccagga ggagttctac
     3061 aagttcatca agcccatcct ggagaagatg gacggcaccg aggagctgct ggtgaagctg
     3121 aaccgcgagg acctgctgcg caagcagcgc accttcgaca acggcagcat cccccaccag
     3181 atccacctgg gcgagctgca cgccatcctg cgccgccagg aggacttcta ccccttcctg
     3241 aaggacaacc gcgagaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     3301 cccctggccc gcggcaacag ccgcttcgcc tggatgaccc gcaagagcga ggagaccatc
     3361 accccctgga acttcgagga ggtggtggac aagggcgcca gcgcccagag cttcatcgag
     3421 cgcatgacca acttcgacaa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     3481 ctgtacgagt acttcaccgt gtacaacgag ctgaccaagg tgaagtacgt gaccgagggc
     3541 atgcgcaagc ccgccttcct gagcggcgag cagaagaagg ccatcgtgga cctgctgttc
     3601 aagaccaacc gcaaggtgac cgtgaagcag ctgaaggagg actacttcaa gaagatcgag
     3661 tgcttcgaca gcgtggagat cagcggcgtg gaggaccgct tcaacgccag cctgggcacc
     3721 taccacgacc tgctgaagat catcaaggac aaggacttcc tggacaacga ggagaacgag
     3781 gacatcctgg aggacatcgt gctgaccctg accctgttcg aggaccgcga gatgatcgag
     3841 gagcgcctga agacctacgc ccacctgttc gacgacaagg tgatgaagca gctgaagcgc
     3901 cgccgctaca ccggctgggg ccgcctgagc cgcaagctta tcaacggcat ccgcgacaag
     3961 cagagcggca agaccatcct ggacttcctg aagagcgacg gcttcgccaa ccgcaacttc
     4021 atgcagctga tccacgacga cagcctgacc ttcaaggagg acatccagaa ggcccaggtg
     4081 agcggccagg gcgacagcct gcacgagcac atcgccaacc tggccggcag ccccgccatc
     4141 aagaagggca tcctgcagac cgtgaaggtg gtggacgagc tggtgaaggt gatgggccgc
     4201 cacaagcccg agaacatcgt gatcgagatg gcccgcgaga accagaccac ccagaagggc
     4261 cagaagaaca gccgcgagcg catgaagcgc atcgaggagg gcatcaagga gctgggcagc
     4321 cagatcctga aggagcaccc cgtggagaac acccagctgc agaacgagaa gctgtacctg
     4381 tactacctgc agaacggccg cgacatgtac gtggaccagg agctggacat caaccgcctg
     4441 agcgactacg acgtggacca catcgtgccc cagagcttcc tgaaggacga cagcatcgac
     4501 aacaaggtgc tgacccgcag cgacaagaac cgcggcaaga gcgacaacgt gcccagcgag
     4561 gaggtggtga agaagatgaa gaactactgg cgccagctgc tgaacgccaa gctgatcacc
     4621 cagcgcaagt tcgacaacct gaccaaggcc gagcgcggcg gcctgagcga gctggacaag
     4681 gccggcttca tcaagcgcca gctggtggag acccgccaga tcaccaagca cgtggcccag
     4741 atcctggaca gccgcatgaa caccaagtac gacgagaacg acaagctgat ccgcgaggtg
     4801 aaggtgatca ccctgaagag caagctggtg agcgacttcc gcaaggactt ccagttctac
     4861 aaggtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtggtg
     4921 ggcaccgccc tgatcaagaa gtaccccaag ctggagagcg agttcgtgta cggcgactac
     4981 aaggtgtacg acgtgcgcaa gatgatcgcc aagagcgagc aggagatcgg caaggccacc
     5041 gccaagtact tcttctacag caacatcatg aacttcttca agaccgagat caccctggcc
     5101 aacggcgaga tccgcaagcg ccccctgatc gagaccaacg gcgagaccgg cgagatcgtg
     5161 tgggacaagg gccgcgactt cgccaccgtg cgcaaggtgc tgagcatgcc ccaggtgaac
     5221 atcgtgaaga agaccgaggt gcagaccggc ggcttcagca aggagagcat cctgcccaag
     5281 cgcaacagcg acaagctgat cgcccgcaag aaggactggg accccaagaa gtacggcggc
     5341 ttcgacagcc ccaccgtggc ctacagcgtg ctggtggtgg ccaaggtgga gaagggcaag
     5401 agcaagaagc tgaagagcgt gaaggagctg ctgggcatca ccatcatgga gcgcagcagc
     5461 ttcgagaaga accccatcga cttcctggag gccaagggct acaaggaggt gaagaaggac
     5521 ctgatcatca agctgcccaa gtacagcctg ttcgagctgg agaacggccg caagcgcatg
     5581 ctggccagcg ccggcgagct gcagaagggc aacgagctgg ccctgcccag caagtacgtg
     5641 aacttcctgt acctggccag ccactacgag aagctgaagg gcagccccga ggacaacgag
     5701 cagaagcagc tgttcgtgga gcagcacaag cactacctgg acgagatcat cgagcagatc
     5761 agcgagttca gcaagcgcgt gatcctggcc gacgccaacc tggacaaggt gctgagcgcc
     5821 tacaacaagc accgcgacaa gcccatccgc gagcaggccg agaacatcat ccacctgttc
     5881 accctgacca acctgggcgc ccccgccgcc ttcaagtact tcgacaccac catcgaccgc
     5941 aagcgctaca ccagcaccaa ggaggtgctg gacgccaccc tgatccacca gagcatcacc
     6001 ggtctgtacg agacccgcat cgacctgagc cagctgggcg gcgacggcgg ctccggacct
     6061 ccaaagaaaa agagaaaagt atacccctac gacgtgcccg actacgccta ataactcgag
     6121 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     6181 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     6241 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     6301 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     6361 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     6421 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     6481 tccattttcg gatctgatcg gcgcgccata ccggtcgcca ccatggtgag caagggcgag
     6541 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     6601 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     6661 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     6721 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     6781 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     6841 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
     6901 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     6961 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     7021 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     7081 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     7141 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     7201 tccaccggcg gcatggacga gctgtacaag taggcggccg cgatcccgcc cctctccctc
     7261 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
     7321 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
     7381 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
     7441 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt
     7501 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     7561 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     7621 gatagttgtg gaaagagtca aatggctcac ctcaagcgta ttcaacaagg ggctgaagga
     7681 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac
     7741 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt
     7801 cctttgaaaa acacgatgat aagcttgcca caacccgcga taattcctgc agccaatatg
     7861 ggatcggcca ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     7921 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     7981 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     8041 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     8101 gcagtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     8161 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     8221 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     8281 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     8341 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgtatg
     8401 cccgacggcg atgatctcgt cgtgactcat ggcgatgcct gcttgccgaa tatcatggtg
     8461 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     8521 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     8581 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     8641 cttcttgacg agttcttctg aacgcggtgc tacgagattt cgattccacc gccgccttct
     8701 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     8761 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     8821 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     8881 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     8941 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     9001 caattccaca caacatacga gccggaagca taaaggtaag cctgaatatt gaaaaaggaa
     9061 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     9121 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     9181 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     9241 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     9301 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     9361 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     9421 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     9481 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     9541 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     9601 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     9661 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     9721 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     9781 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     9841 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     9901 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     9961 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
    10021 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
    10081 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
    10141 aaaaccaccg ctaccagcgg tggttttttt gccggatcaa gagctaccaa ctctttttcc
    10201 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
    10261 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
    10321 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
    10381 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
    10441 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
    10501 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
    10561 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
    10621 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
    10681 gaaaaacgcc agca
//