LOCUS       sequence-393001-       12971 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible Piggybac vector expressing SpCas9 with Blasticidin 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12971)
  AUTHORS   Bajpai VK, Swigut T, Mohammed J, Naqvi S, Arreola M, Tycko J, Kim 
            TC, Pritchard JK, Bassik MC, Wysocka J
  TITLE     A genome-wide genetic screen uncovers determinants of human 
            pigmentation.
  JOURNAL   Science. 2023 Aug 11;381(6658):eade6289. doi: 
            10.1126/science.ade6289. Epub 2023 Aug 11.
  PUBMED    37561850
REFERENCE   2  (bases 1 to 12971)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science."; 
            date: "2023-08-11"; volume: "381(6658):eade6289. doi"; pages: " 
            10.1126/science.ade6289. Epub 2023 Aug 11"
COMMENT     Sequence Label: Pb-TRE3G-SpCas9-IRES-blast
FEATURES             Location/Qualifiers
     source          1..12971
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1482..1516
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    2279..2400
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2290..2309)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2344..2363
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2526..2924)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    2931..3503
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3090..3109)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     3319..3336
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     protein_bind    3526..3559
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     CDS             complement(3560..4267)
                     /codon_start=1
                     /product="reverse tetracycline transactivator 3"
                     /label=rtTA3
                     /note="activates expression from Tet-responsive promoters 
                     in the presence of doxycycline"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPG"
     protein_bind    4274..4307
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     promoter        complement(4320..5531)
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     complement(4464..4483)
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     misc_feature    5539..6127
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             5645..5656
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(6158..10261)
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(6161..10261)
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(10262..10282)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(10300..10324)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(10321..10359)
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    10371..10389
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10407..10425
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10442..10460
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10478..10496
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10514..10532
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10549..10567
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     repeat_region   11005..11067
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11197..11214)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11197..11213)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11206..11228)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11355..11810
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11442..11461)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11652..11673
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11836..11940
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11941..12009,12010..12801)
                     /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(12159..12178)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg cccgccgggt
     1021 aactcacggg gtatccatgt ccatttctgc ggcatccagc caggataccc gtcctcgctg
     1081 acgtaatatc ccagcgccgc accgctgtca ttaatctgca caccggcacg gcagttccgg
     1141 ctgtcgccgg tattgttcgg gttgctgatg cgcttcgggc tgaccatccg gaactgtgtc
     1201 cggaaaagcc gcgacgaact ggtatcccag gtggcctgaa cgaacagttc accgttaaag
     1261 gcgtgcatgg ccacaccttc ccgaatcatc atggtaaacg tgcgttttcg ctcaacgtca
     1321 atgcagcagc agtcatcctc ggcaaactct ttccatgccg cttcaacctc gcgggaaaag
     1381 gcacgggctt cttcctcccc gatgcccaga tagcgccagc ttgggcgatg actgagccgg
     1441 aaaaaagacc cgacgatatg atcctgatgc agctagatta accctagaaa gatagtctgc
     1501 gtaaaattga cgcatgcatt cttgaaatat tgctctctct ttctaaatag cgcgaatccg
     1561 tcgctgtgca tttaggacat ctcagtcgcc gcttggagct cccgtgaggc gtgcttgtca
     1621 atgcggtaag tgtcactgat tttgaactat aacgaccgcg tgagtcaaaa tgacgcatga
     1681 ttatctttta cgtgactttt aagatttaac tcatacgata attatattgt tatttcatgt
     1741 tctacttacg tgataactta ttatatatat attttcttgt tatagatatc agggacagcc
     1801 cccccccaaa gcccccaggg atgtaattac gtccctcccc cgctaggggg cagcagcgag
     1861 ccgcccgggg ctccgctccg gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg
     1921 gggacagccc gggcacgggg aaggtggcac gggatcgctt tcctctgaac gcttctcgct
     1981 gctctttgag cctgcagaca cctgggggga tacggggaaa aagccatacc aatgggccct
     2041 aaaaaaggaa tccagtcaat tccggggcta aacctggctg ccactgtttc tttagggact
     2101 tcgttcctgt gaggacagga aaccctgcaa cctggttgac cagagcaagg gatataagaa
     2161 cccagtttgg gaacactgct gtttaaatat taaacaggga tgtcttgtcc cccggaaggg
     2221 agaccagatg agcttctgat agactaattg acatcatttg agtcaattcc agacatgata
     2281 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     2341 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     2401 tagttagtta acgcatgata caaaggcatt aaagcagcgt atccacatag cgtaaaagga
     2461 gcaacatagt taagaatacc agtcaatctt tcacaaattt tgtaatccag aggttgattg
     2521 tcgacttagc cctcccacac ataaccagag ggcagcaatt cacgaatccc aactgccgtc
     2581 ggctgtccat cactgtcctt cactatggct ttgatcccag gatgcagatc gagaagcacc
     2641 tgtcggcacc gtccgcaggg gctcaagatg cccctgttct catttccgat cgcgacgata
     2701 caagtcaggt tgccagctgc cgcagcagca gcagtgccca gcaccacgag ttctgcacaa
     2761 ggtcccccag taaaatgata tacattgaca ccagtgaaga tgcggccgtc gctagagaga
     2821 gctgcgctgg cgacgctgta gtcttcagag atggggatgc tgttgattgt agccgttgct
     2881 ctttcaatga gggtggattc ttcttgagac aaaggcttgg ccatccatgg tattatcgtg
     2941 tttttcaaag gaaaaccacg tccccgtggt tcggggggcc tagacgtttt tttaacctcg
     3001 actaaacaca tgtaaagcat gtgcaccgag gccccagatc agatcccata caatggggta
     3061 ccttctgggc atccttcagc cccttgttga atacgcttga ggagagccat ttgactcttt
     3121 ccacaactat ccaactcaca acgtggcact ggggttgtgc cgcctttgca ggtgtatctt
     3181 atacacgtgg cttttggccg cagaggcacc tgtcgccagg tggggggttc cgctgcctgc
     3241 aaagggtcgc tacagacgtt gtttgtcttc aagaagcttc cagaggaact gcttccttca
     3301 cgacattcaa cagaccttgc attcctttgg cgagagggga aagaccccta ggaatgctcg
     3361 tcaagaagac agggccaggt ttccgggccc tcacattgcc aaaagacggc aatatggtgg
     3421 aaaataacat atagacaaac gcacaccggc cttattccaa gcggcttcgg ccagtaacgt
     3481 tagggggggg ggagggagag gggcggaatt tgcggccgcg gatccataac ttcgtataat
     3541 gtatactata cgaagttatt tacccgggga gcatgtcaag gtcaaaatcg tcaagggcat
     3601 cggctgggag catgtctaag tcaaaatcgt caagggcgtc ggtcggcccg ccgctttcgc
     3661 actttagctg tttctccagg ccacatatga ttagttccag gccgaaaagg aaggcaggtt
     3721 cggctccctg ccggtcgaac agctcaattg cttgtctcag aagtgggggc atagaatcgg
     3781 tggtaggtgt ctctctttcc tcttttgcta cttgatgctc ctgttcctcc aatacgcagc
     3841 ccagtgtaaa gtggcccacg gcggacagag cgtacagtgc gttctccagg gagaagcctt
     3901 gctgacacag gaacgcgagc tgattttcca gggtttcgta ctgtttctct gttgggcggg
     3961 tgccgagatg cactttagcc ccgtcgcgat gtgagaggag agcacagcgg tatgacttgg
     4021 cgttgttccg cagaaagtct tgccatgact cgccttccag ggggcagaag tgggtatgat
     4081 gcctgtccag catctcgatt ggcagggcat cgagcagggc ccgcttgttc ttcacgtgcc
     4141 agtacagggt aggctgctca actcccagct tttgagcgag tttccttgtc gtcaggcctt
     4201 cgataccgac accattgagt aattccagag ctccgtttat gactttgctc ttgtccagcc
     4261 tagacatggt ggcataactt cgtataatgt atactatacg aagttatgga tccgctagcg
     4321 tctaacaaaa aagccaaaaa cggccagaat ttagcggaca atttactagt ctaacactga
     4381 aaattacata ttgacccaaa tgattacatt tcaaaaggtg cctaaaaaac ttcacaaaac
     4441 acactcgcca accccgagcg catagttcaa aaccggagct tcagctactt aagaagatag
     4501 gtacataaaa ccgaccaaag aaactgacgc ctcacttatc cctcccctca ccagaggtcc
     4561 ggcgcctgtc gattcaggag agcctaccct aggcccgaac cctgcgtcct gcgacggaga
     4621 aaagcctacc gcacacctac cggcaggtgg ccccaccctg cattataagc caacagaacg
     4681 ggtgacgtca cgacacgacg agggcgcgcg ctcccaaagg tacgggtgca ctgcccaacg
     4741 gcaccgccat aactgccgcc cccgcaacag acgacaaacc gagttctcca gtcagtgaca
     4801 aacttcacgt cagggtcccc agatggtgcc ccagcccatc tcacccgaat aagagctttc
     4861 ccgcattagc gaaggcctca agaccttggg ttcttgccgc ccaccatgcc ccccaccttg
     4921 tttcaacgac ctcacagccc gcctcacaag cgtcttccat tcaagactcg ggaacagccg
     4981 ccattttgct gcgctccccc caacccccag ttcagggcaa ccttgctcgc ggacccagac
     5041 tacagccctt ggcggtctct ccacacgctt ccgtcccacc gagcggcccg gcggccacga
     5101 aagccccggc cagcccagca gcccgctact caccaagtga cgatcacagc gatccacaaa
     5161 caagaaccgc gacccaaatc ccggctgcga cggaactagc tgtgccacac ccggcgcgtc
     5221 cttatataat catcggcgtt caccgcccca cggagatccc tccgcagaat cgccgagaag
     5281 ggactacttt tcctcgcctg ttccgctctc tggaaagaaa accagtgccc tagagtcacc
     5341 caagtcccgt cctaaaatgt ccttctgctg atactggggt tctaaggccg agtcttatga
     5401 gcagcgggcc gctgtcctga gcgtccgggc ggaaggatca ggacgctcgc tgcgcccttc
     5461 gtctgacgtg gcagcgctcg ccgtgaggag gggggcgccc gcgggaggcg ccaaaacccg
     5521 gcgcggaggc caacgcgtgc ggggaggcgg cccaaaggga gatccgactc gtctgagggc
     5581 gaaggcgaag acgcggaaga ggccgcagag ccggcagcag gccgcgggaa ggaaggtccg
     5641 ctggattgag ggccgaaggg acgtagcaga aggacgtccc gcgcagaatc caggtggcaa
     5701 cacaggcgag cagccatgga aaggacgtca gcttccccga caacaccacg gaattgtcag
     5761 tgcccaacag ccgagcccct gtccagcagc gggcaaggca ggcggcgatg agttccgccg
     5821 tggcaatagg gagggggaaa gcgaaagtcc cggaaaggag ctgacaggtg gtggcaatgc
     5881 cccaaccagt gggggttgcg tcagcaaaca cagtgcacac cacgccacgt tgcctgacaa
     5941 cgggccacaa ctcctcataa agagacagca accaggattt atacaaggag gagaaaatga
     6001 aagccatacg ggaagcaata gcatgataca aaggcattaa agcagcgtat ccacatagcg
     6061 taaaaggagc aacatagtta agaataccag tcaatctttc acaaattttg taatccagag
     6121 gttgattgtc gatagatctc gagcccgggc ggaattctta gtcgcctccc agctgagaca
     6181 ggtcgatccg tgtctcgtac aggccggtga tgctctggtg gatcagggtg gcgtccagca
     6241 cctctttggt gctggtgtac ctcttccggt cgatggtggt gtcaaagtac ttgaaggcgg
     6301 caggggctcc cagattggtc agggtaaaca ggtggatgat attctcggcc tgctctctga
     6361 tgggcttatc ccggtgcttg ttgtaggcgg acagcacttt gtccagatta gcgtcggcca
     6421 ggatcactct cttggagaac tcgctgatct gctcgatgat ctcgtccagg tagtgcttgt
     6481 gctgttccac aaacagctgt ttctgctcat tatcctcggg ggagcccttc agcttctcat
     6541 agtggctggc caggtacagg aagttcacat atttggaggg cagggccagt tcgtttccct
     6601 tctgcagttc gccggcagag gccagcattc tcttccggcc gttttccagc tcgaacaggg
     6661 agtacttagg cagcttgatg atcaggtcct ttttcacttc tttgtagccc ttggcttcca
     6721 gaaagtcgat gggattcttc tcgaagctgc ttctttccat gatggtgatc cccagcagct
     6781 ctttcacact cttcagtttc ttggacttgc ccttttccac tttggccacc accagcacag
     6841 aataggccac ggtggggctg tcgaagccgc cgtacttctt agggtcccag tccttctttc
     6901 tggcgatcag cttatcgctg ttcctcttgg gcaggataga ctctttgctg aagccgcctg
     6961 tctgcacctc ggtctttttc acgatattca cttggggcat gctcagcact ttccgcacgg
     7021 tggcaaaatc ccggccctta tcccacacga tctccccggt ttcgccgttt gtctcgatca
     7081 gaggccgctt ccggatctcg ccgttggcca gggtaatctc ggtcttgaaa aagttcatga
     7141 tgttgctgta gaagaagtac ttggcggtag ccttgccgat ttcctgctcg ctcttggcga
     7201 tcatcttccg cacgtcgtac accttgtagt cgccgtacac gaactcgctt tccagcttag
     7261 ggtacttttt gatcagggcg gttcccacga cggcgttcag gtaggcgtcg tgggcgtggt
     7321 ggtagttgtt gatctcgcgc actttgtaaa actggaaatc cttccggaaa tcggacacca
     7381 gcttggactt cagggtgatc actttcactt cccggatcag cttgtcattc tcgtcgtact
     7441 tagtgttcat ccgggagtcc aggatctgtg ccacgtgctt tgtgatctgc cgggtttcca
     7501 ccagctgtct cttgatgaag ccggccttat ccagttcgct caggccgcct ctctcggcct
     7561 tggtcagatt gtcgaacttt ctctgggtaa tcagcttggc gttcagcagc tgccgccagt
     7621 agttcttcat cttcttcacg acctcttcgg agggcacgtt gtcgctcttg ccccggttct
     7681 tgtcgcttct ggtcagcacc ttgttgtcga tggagtcgtc cttcagaaag ctctgaggca
     7741 cgatatggtc cacatcgtag tcggacagcc ggttgatgtc cagttcctgg tccacgtaca
     7801 tatcccgccc attctgcagg tagtacaggt acagcttctc gttctgcagc tgggtgtttt
     7861 ccacggggtg ttctttcagg atctggctgc ccagctcttt gatgccctct tcgatccgct
     7921 tcattctctc gcggctgttc ttctgtccct tctgggtggt ctggttctct ctggccattt
     7981 cgatcacgat gttctcgggc ttgtgccggc ccatcacttt cacgagctcg tccaccacct
     8041 tcactgtctg caggatgccc ttcttaatgg cggggctgcc ggccagattg gcaatgtgct
     8101 cgtgcaggct atcgccctgg ccggacacct gggctttctg gatgtcctct ttaaaggtca
     8161 ggctgtcgtc gtggatcagc tgcatgaagt ttctgttggc gaagccgtcg gacttcagga
     8221 aatccaggat tgtcttgccg gactgcttgt cccggatgcc gttgatcagc ttccggctca
     8281 gcctgcccca gccggtgtat ctccgccgct tcagctgctt catcactttg tcgtcgaaca
     8341 ggtgggcata ggttttcagc cgttcctcga tcatctctct gtcctcaaac agtgtcaggg
     8401 tcagcacgat atcttccaga atgtcctcgt tttcctcatt gtccaggaag tccttgtcct
     8461 tgataatttt cagcagatcg tggtatgtgc ccagggaggc gttgaaccga tcttccacgc
     8521 cggagatttc cacggagtcg aagcactcga ttttcttgaa gtagtcctct ttcagctgct
     8581 tcacggtcac tttccggttg gtcttgaaca gcaggtccac gatggccttt ttctgctcgc
     8641 cgctcaggaa ggcgggcttt ctcattccct cggtcacgta tttcactttg gtcagctcgt
     8701 tatacacggt gaagtactcg tacagcaggc tgtgcttggg cagcaccttc tcgttgggca
     8761 ggttcttatc gaagttggtc atccgctcga tgaagctctg ggcggaagcg cccttgtcca
     8821 ccacttcctc gaagttccag ggggtgatgg tttcctcgct ctttctggtc atccaggcga
     8881 atctgctgtt tcccctggcc agagggccca cgtagtaggg gatgcggaag gtcaggatct
     8941 tctcgatctt ttcccggttg tccttcagga atgggtaaaa atcttcctgc cgccgcagaa
     9001 tggcgtgcag ctctcccagg tggatctggt gggggatgct gccgttgtcg aaggtccgct
     9061 gcttccgcag caggtcctct ctgttcagct tcacgagcag ttcctcggtg ccgtccatct
     9121 tttccaggat gggcttgatg aacttgtaga actcttcctg gctggctccg ccgtcaatgt
     9181 agccggcgta gccgttcttg ctctggtcga agaaaatctc tttgtacttc tcaggcagct
     9241 gctgccgcac gagagctttc agcagggtca ggtcctggtg gtgctcgtcg tatctcttga
     9301 tcatagaggc gctcaggggg gccttggtga tctcggtgtt cactctcagg atgtcgctca
     9361 gcaggatggc gtcggacagg ttcttggcgg ccagaaacag gtcggcgtac tggtcgccga
     9421 tctgggccag caggttgtcc aggtcgtcgt cgtaggtgtc cttgctcagc tgcagtttgg
     9481 catcctcggc caggtcgaag ttgctcttga agttgggggt caggcccagg ctcagggcaa
     9541 tcaggtttcc gaacaggcca ttcttcttct cgccgggcag ctgggcgatc agattttcca
     9601 gccgtctgct cttgctcagt ctggcagaca ggatggcctt ggcgtccacg ccgctggcgt
     9661 tgatggggtt ttcctcgaac agctggttgt aggtctgcac cagctggatg aacagcttgt
     9721 ccacgtcgct gttgtcgggg ttcaggtcgc cctcgatcag gaagtggccc cggaacttga
     9781 tcatgtgggc cagggccaga tagatcagcc gcaggtcggc cttgtcggtg ctgtccacca
     9841 gtttctttct caggtggtag atggtggggt acttctcgtg gtaggccacc tcgtccacga
     9901 tgttgccgaa gatggggtgc cgctcgtgct tcttatcctc ttccaccagg aaggactctt
     9961 ccagtctgtg gaagaagctg tcgtccacct tggccatctc gttgctgaag atctcttgca
    10021 gatagcagat ccggttcttc cgtctggtgt atcttcttct ggcggttctc ttcagccggg
    10081 tggcctcggc tgtttcgccg ctgtcgaaca gcagggctcc gatcaggttc ttcttgatgc
    10141 tgtgccggtc ggtgttgccc agcaccttga atttcttgct gggcaccttg tactcgtcgg
    10201 tgatcacggc ccagcccaca gagttggtgc cgatgtccag gccgatgctg tacttcttgt
    10261 ccactttcct cttctttttg gggctagcca tgccggtgcg atctgacggt tcactaaacg
    10321 agctctgctt atataggcct cccaccgtac acgcctacct cgacatacgt tctctatcac
    10381 tgatagggag taaactcgac atacgttctc tatcactgat agggataaac tcgacatacg
    10441 ttctctatca ctgataggga gtaaactcga catacgttct ctatcactga tagggagtaa
    10501 actcgacata cgttctctat cactgatagg gagtaaactc gacatcgttc tctatcactg
    10561 atagggagta aactcgtcta gtctggcctt ggaggccttt tccccgtatc cccccaggtg
    10621 tctgcaggct caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc
    10681 ccgtgcccgg gctgtccccg cacgctgccg gctcggggat gcggggggag cgccggaccg
    10741 gagcggagcc ccgggcggct cgctgctgcc ccctagcggg ggagggacgt aattacatcc
    10801 ctgggggctt tggggggggg ctgtccctca taaaagtttt gttactttat agaagaaatt
    10861 ttgagttttt gtttttttta ataaataaat aaacataaat aaattgtttg ttgaatttat
    10921 tattagtatg taagtgtaaa tataataaaa cttaatatct attcaaatta ataaataaac
    10981 ctcgatatac agaccgataa aacacatgcg tcaattttac acatgattat ctttaacgta
    11041 cgtcacaata tgattatctt tctagggtta atctagctgc gtgttctgca gcgtgtcgag
    11101 catcttcatc tgctccatca cgctgtaaaa cacatttgca ccgcgagtct gcccgtcctc
    11161 cacgggttca aaaacgtgaa tgaacgaggc gcgctcactg gccgtcgttt tacaacgtcg
    11221 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
    11281 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
    11341 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
    11401 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
    11461 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
    11521 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
    11581 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
    11641 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
    11701 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
    11761 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
    11821 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
    11881 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
    11941 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
    12001 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
    12061 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
    12121 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
    12181 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
    12241 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
    12301 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
    12361 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
    12421 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
    12481 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
    12541 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
    12601 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
    12661 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
    12721 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
    12781 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
    12841 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
    12901 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
    12961 aaaggatctt c
//
