LOCUS       sequence-365380-       14831 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Big-IN positive control payload encoding EF1a-driven GFP-T2A-BSD. 
            Harbors a backbone counterselectable TK cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14831)
  AUTHORS   Brosh R, Laurent JM, Ordonez R, Huang E, Hogan MS, Hitchcock AM, 
            Mitchell LA, Pinglay S, Cadley JA, Luther RD, Truong DM, Boeke JD, 
            Maurano MT
  TITLE     A versatile platform for locus-scale genome rewriting and 
            verification.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 Mar 9;118(10). pii: 2023952118. doi: 
            10.1073/pnas.2023952118.
  PUBMED    33649239
REFERENCE   2  (bases 1 to 14831)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2021-03-9"; volume: "118(10). pii: 
            2023952118. doi"; pages: " 10.1073/pnas.2023952118"
COMMENT     Sequence Label: pPL001-BBTK
FEATURES             Location/Qualifiers
     source          1..14831
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(398..1369)
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     CDS             complement(1369..2544)
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     misc_feature    2870..3120
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(3123..3878)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      3969..4188
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     rep_origin      4256..4787
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      4264..4878
                     /label=oriV
                     /note="origin of replication for the bacterial F plasmid"
     misc_feature    5729..6232
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     mobile_element  6234..7001
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     promoter        7029..7131
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             7132..7947
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             7132..7947
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(7205..7224)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(8524..9618)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     protein_bind    10099..10132
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    10133..10340
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     10329..10346
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(10374..10772)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(10773..10826)
                     /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             complement(join(10836..11546,11547..11549,11550..11552))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(10836..11546,11547..11549,11550..11552))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     11227..11246
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     11486..11507
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(11573..12751)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          11582..12520
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(11605..11625)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    12752..12785
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     polyA_signal    13020..13141
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13031..13050)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     13085..13104
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(13142..14137)
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRSMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     promoter        complement(14143..14626)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(14179..14197)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
ORIGIN
        1 ggtcccagac cgataatcag accgacaaca cgagtgggat cgtggtccca gactaataat
       61 cagaccgacg atacgagtgg gaccgtggtc ccagactaat aatcagaccg acgatacgag
      121 tgggaccgtg gtyccagact aataatcaga ccgacgatac gagtgggacc gtggtcccag
      181 actaataatc agaccgacga tacgagtggg accgtggtcc cagactaata atcagaccga
      241 cgatacgagt gggaccgtgg tcccagtctg attatcagac cgacgataca agtggaacag
      301 tgggcccaga gagaatattc aggccagtta tgctttctgg cctgtaacaa aggacattaa
      361 gtaaagacag ataaacgtag actaaaacga ggtcgcatca gggtgctggc ttttcaagtt
      421 ccttaagaat ggcctcaatt ttctctatac actcagttgg aacacgggac ctgtccaggt
      481 taagcaccat tttatcgccc ttatacaata ctgtcgctcc aggagcaaac tgatgtcgtg
      541 agcttaaact agttcttgat gcagatgacg ttttaagcac agaagttaaa agagtgataa
      601 cttcttcagc ttcaaatatc accccagctt ttttctgctc atgaaggtta gatgcctgct
      661 gcttaagtaa ttcctcttta tctgtaaagg ctttttgaag tgcatcacct gaccgggcag
      721 atagttcacc ggggtgagaa aaaagagcaa caactgattt aggcaatttg gcggtgttga
      781 tacagcgggt aataatctta cgtgaaatat tttccgcatc agccagcgca gaaatatttc
      841 cagcaaattc attctgcaat cggcttgcat aacgctgacc acgttcataa gcacttgttg
      901 ggcgataatc gttacccaat ctggataatg cagccatctg ctcatcatcc agctcgccaa
      961 ccagaacacg ataatcactt tcggtaagtg cagcagcttt acgacggcga ctcccatcgg
     1021 caatttctat gacaccagat actcttcgac cgaacgccgg tgtctgttga ccagtcagta
     1081 gaaaagaagg gatgagatca tccagtgcgt cctcagtaag cagctcctgg tcacgttcat
     1141 tacctgacca tacccgagag gtcttctcaa cactatcacc ccggagcact tcaagagtaa
     1201 acttcacatc ccgaccacat acaggcaaag taatggcatt accgcgagcc attactccta
     1261 cgcgcgcaat taacgaatcc accatcgggg cagctggtgt cgataacgaa gtatcttcaa
     1321 ccggttgagt attgagcgta tgttttggaa taacaggcgc acgcttcatt atctaatctc
     1381 ccagcgtggt ttaatcagac gatcgaaaat ttcattgcag acaggttccc aaatagaaag
     1441 agcatttctc caggcaccag ttgaagagcg ttgatcaatg gcctgttcaa aaacagttct
     1501 catccggatc tgacctttac caacttcatc cgtttcacgt acaacatttt ttagaaccat
     1561 gcttccccag gcatcccgaa tttgctcctc catccacggg gactgagagc cattgctatt
     1621 gctgtatttg gtaagcaaaa tacgtacatc aggctcgaac cctttaagat caacgttctt
     1681 gagcagatca cgaagcatat cgaaaaactg cagtgcggag gtgtagtcaa acaactcagc
     1741 aggcgtggga acaatcagca catcagcagc acatacgaca ttaatcgtgc cgatacccag
     1801 gttaggcgcg ctgtcaataa ctatgacatc atagtcatga gcaacagttt caatggccag
     1861 tcggagcatc aggtgtggat cggtgggcag tttaccttca tcaaatttgc ccattaactc
     1921 agtttcaata cggtgcagag ccagacagga aggaataatg tcaagccccg gccagcaagt
     1981 gggctttatt gcataagtga catcgtcctt ttccccaaga tagaaaggca ggagagtgtc
     2041 ttctgcatga atatgaagat ctggtaccca tccgtgatac attgaggctg ttccctgggg
     2101 gtcgttacct tccacgagca aaacacgtag ccccttcaga gccagatcct gagcaagatg
     2161 aacagaaact gaggttttgt aaacgccacc tttatgggca gcaaccccga tcaccggtgg
     2221 aaatacgtct tcagcacgtc gcaatcgcgt accaaacaca tcacgcatat gattaatttg
     2281 ttcaattgta taaccaacac gttgctcaac ccgtcctcga atttccatat ccgggtgcgg
     2341 tagtcgccct gctttctcgg catctctgat agcctgagaa gaaaccccaa ctaaatccgc
     2401 tgcttcacct attctccagc gccgggttat tttcctcgct tccgggctgt catcattaaa
     2461 ctgtgcaatg gcgatagcct tcgtcatttc atgaccagcg tttatgcact ggttaagtgt
     2521 ttccatgagt ttcattctga acatccttta atcattgctt tgcgtttttt tattaaatct
     2581 tgcaatttac tgcaaagcaa caacaaaatc gcaaagtcat caaaaaaccg caaagttgtt
     2641 taaaataaga gcaacactac aaaaggagat aagaagagca catacctcag tcacttatta
     2701 tcactagcgc tcgccgcagc cgtgtaaccg agcatagcga gcgaactggc gaggaagcaa
     2761 agaagaactg ttctgtcaga tagctcttac gctcagcgca agaagaaata tccaccgtgg
     2821 gaaaaactcc aggtagaggt acacacgcgg atagccaatt cagagtaata aactgtgata
     2881 atcaaccctc atcaatgatg acgaactaac ccccgatatc aggtcacatg acgaagggaa
     2941 agagaaggaa atcaactgtg acaaactgcc ctcaaatttg gcttccttaa aaattacagt
     3001 tcaaaaagta tgagaaaatc catgcaggct gaaggaaaca gcaaaactgt gacaaattac
     3061 cctcagtagg tcagaacaaa tgtgacgaac caccctcaaa tctgtgacag ataaccctca
     3121 gactatcctg tcgtcatgga agtgatatcg cggaaggaaa atacgatatg agtcgtctgg
     3181 cggcctttct ttttctcaat gtatgagagg cgcattggag ttctgctgtt gatctcatta
     3241 acacagacct gcaggaagcg gcggcggaag tcaggcatac gctggtaact ttgaggcagc
     3301 tggtaacgct ctatgatcca gtcgattttc agagagacaa tgcctgagcc atccggctta
     3361 cgatactgac acagggattc gtataaacgc atggcatacg gattggtgat ttcttttgtt
     3421 tcactaagcc gaaactgcgt aaaccggttc tgtaacccga taaagaaggg aatgagatat
     3481 gggttgatat gtacactgta aagccctctg gatggactgt gcgcacgttt gataaaccaa
     3541 ggaaaagatt catagccttt ttcatcgccg gcatcctctt cagggcgata aaaaaccact
     3601 tccttccccg cgaaactctt caatgcctgc cgtatatcct tactggcttc cgcagaggtc
     3661 aatccgaata tttcagcata tttagcaaca tggatctcgc agataccgtc atgttcctgt
     3721 agggtgccat cagattttct gatctggtca acgaacagat acagcatacg tttttgatcc
     3781 cgggagagac tatatgccgc ctcagtgagg tcgtttgact ggacgattcg cgggctattt
     3841 ttacgtttct tgtgattgat aaccgctgtt tccgccatga cagatccatg tgaagtgtga
     3901 caagttttta gattgtcaca ctaaataaaa aagagtcaat aagcagggat aactttgtga
     3961 aaaaacagct tcttctgagg gcaatttgtc acagggttaa gggcaatttg tcacagacag
     4021 gactgtcatt tgagggtgat ttgtcacact gaaagggcaa tttgtcacaa caccttctct
     4081 agaaccagca tggataaagg cctacaaggc gctctaaaaa agaagatcta aaaactataa
     4141 aaaaaataat tataaaaata tccccgtgga taagtggata accccaaggg aagttttttc
     4201 aggcatcgtg tgtaagcaga atatataagt gctgttccct ggtgcttcct cgctcactcg
     4261 accgggaggg ttcgagaagg gggggcaccc cccttcggcg tgcgcggtca cgcgcacagg
     4321 gcgcagccct ggttaaaaac aaggtttata aatattggtt taaaagcagg ttaaaagaca
     4381 ggttagcggt ggccgaaaaa cgggcggaaa cccttgcaaa tgctggattt tctgcctgtg
     4441 gacagcccct caaatgtcaa taggtgcgcc cctcatctgt cagcactctg cccctcaagt
     4501 gtcaaggatc gcgcccctca tctgtcagta gtcgcgcccc tcaagtgtca ataccgcagg
     4561 gcacttatcc ccaggcttgt ccacatcatc tgtgggaaac tcgcgtaaaa tcaggcgttt
     4621 tcgccgattt gcgaggctgt ccagctccac gtcgccggcc gaaatcgagc ctgcccctca
     4681 tctgtcaacg ccgcgccggg tgagtcggcc cctcaagtgt caacgtccgc ccctcatctg
     4741 tcagtgaggg ccaagttttc cgcgaggtat ccacaacgcc ggcggccggc cgcggtgtct
     4801 cgcacacggc ttcgacggcg tttctggcgc gtttgcaggg ccatagacag ccgccagccc
     4861 agcggcgagg gcaaccagct cgagggcttc gccctgtcgc tcgactgcgg cgagcactac
     4921 tggctgtaaa aggacagacc acatcatggt tctgtgttca ttaggttgtt ctgtccattg
     4981 ctgacataat ccgctccact tcaacgtaac accgcacgaa gatttctatt gttcctgaag
     5041 gcatattcaa atcgttttcg ttaccgcttg caggcatcat gacagaacac tacttcctat
     5101 aaacgctaca caggctcctg agattaataa tgcggatctc tacgataatg ggagattttc
     5161 ccgactgttt cgctcgcttc tcagtggata acagccagct tctctgttta acagacaaaa
     5221 acagcatatc cactcagttc cacatttcca tataaaggcc aaggcattta ttctcaggat
     5281 aattgtttca gcatcgcaac cgcatcagac tccggcatcg caaactgcac ccggtgccgg
     5341 gcagccacat ccagcgcaaa aaccttcgtg tagacttccg ttgaactgat ggacttatgt
     5401 cccatcaggc tttgcagaac tttcagcggt ataccggcat acagcatgtg catcgcatag
     5461 gaatggcgga acgtatgtgg tgtgaccgga acagagaacg tcacaccgtc agcagcagcg
     5521 gcggcaaccg cctccccaat ccaggtcctg accgttctgt ccgtcacttc ccagatccgc
     5581 gctttctctg tccttcctgt gcgacggtta cgccgctcca tgagcttatc gcgaataaat
     5641 acctgtgacg gaagatcact tcgcagaata aataaatcct ggtgtccctg ttgataccgg
     5701 gaagccctgg gccaactttt ggcgaaaaga tcgcttgcct gtaacttaca cgcgcctcgt
     5761 atcttttaat gatggaataa tttgggaatt tactctgtgt ttatttattt ttatgttttg
     5821 tatttggatt ttagaaagta aataaagaag gtagaagagt tacggaatga agaaaaaaaa
     5881 ataaacaaag gtttaaaaaa tttcaacaaa aagcgtactt tacatatata tttattagac
     5941 aagaaaagca gattaaatag atatacattc gattaacgat aagtaaaatg taaaatcaca
     6001 ggattttcgt gtgtggtctt ctacacagac aagatgaaac aattcggcat taatacctga
     6061 gagcaggaag agcaagataa aaggtagtat ttgttggcga tccccctaga gtcttttaca
     6121 tcttcggaaa acaaaaacta ttttttcttt aatttctttt tttactttct atttttaatt
     6181 tatatattta tattaaaaaa tttaaattat aattattttt atagcacgtg atgggtaatg
     6241 actccaactt attgatagtg ttttatgttc agataatgcc cgatgacttt gtcatgcagc
     6301 tccaccgatt ttgagaacga cagcgacttc cgtcccagcc gtgccaggtg ctgcctcaga
     6361 ttcaggttat gccgctcaat tcgctgcgta tatcgcttgc tgattacgtg cagctttccc
     6421 ttcaggcggg attcatacag cggccagcca tccgtcatcc atatcaccac gtcaaagggt
     6481 gacagcaggc tcataagacg ccccagcgtc gccatagtgc gttcaccgaa tacgtgcgca
     6541 acaaccgtct tccggagact gtcatacgcg taaaacagcc agcgctggcg cgatttagcc
     6601 ccgacatagc cccactgttc gtccatttcc gcgcagacga tgacgtcact gcccggctgt
     6661 atgcgcgagg ttaccgactg cggcctgagt tttttaagtg acgtaaaatc gtgttgaggc
     6721 caacgcccat aatgcgggct gttgcccggc atccaacgcc attcatggcc atatcaatga
     6781 ttttctggtg cgtaccgggt tgagaagcgg tgtaagtgaa ctgcagttgc catgttttac
     6841 ggcagtgaga gcagagatag cgctgatgtc cggcggtgct tttgccgtta cgcaccaccc
     6901 cgtcagtagc tgaacaggag ggacagctga tagaaacaga agccactgga gcacctcaaa
     6961 aacaccatca tacactaaat cagtaagttg gcagcatcac ccacgtgatg aaaaggaccc
     7021 agggacgttg atcggcacgt aagaggttcc aactttcacc ataatgaaat aagatcacta
     7081 ccggacgtat tttttgagtt atcgagattt tcaggagcta aggaagctaa aatgagccat
     7141 attcaacggg aaacgtcttg ctcgaggccg cgattaaatt ccaacatgga tgctgattta
     7201 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
     7261 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     7321 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     7381 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga
     7441 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     7501 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     7561 gatcgcgtat ttcgtctggc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     7621 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     7681 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     7741 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     7801 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     7861 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     7921 catttgatgc tcgatgagtt tttctaattt ttttaaggca gttattggtg cccttaaacg
     7981 cctggttgct acgcctgaat aagtgataat aagcggatga atggcagaaa ttcgatgata
     8041 agctgtcaaa catgagaatt gtggctgttg catgtggtgt acagtagatg tttcgcaggg
     8101 ctaagaacgg atctatttcg agtcccccac cttccatcgc ggttactttg atccgcatca
     8161 cgctccggag taattgaaat caacaccatc tgataaaggc tccagtgtgc gttcctcatc
     8221 tcgaccgtcg cgtttctgtt gttgatcggc tgcgtagttg ggcggccgcg gtgccctcct
     8281 ccttgtcaat attaatgtta aagtgcaatt ctttttcctt atcacgttga gccattagta
     8341 tcaatttgct tacctgtatt cctttacatc ctcctttttc tccttcttga taaatgtatg
     8401 tagattgcgt atatagtttc gtctacccta tgaacatatt ccattttgta atttcgtgtc
     8461 gtttctatta tgaatttcat ttataaagtt tatgtacaaa tatcataaaa aaagagaatc
     8521 tttttaagca aggattttct taacttcttc ggcgacagca tcaccgactt cggtggtact
     8581 gttggaacca cctaaatcac cagttctgat acctgcatcc aaaacctttt taactgcatc
     8641 ttcaatggcc ttaccttctt caggcaagtt caatgacaat ttcaacatca ttgcagcaga
     8701 caagatagtg gcgatagggt tgaccttatt ctttggcaaa tctggagcag aaccgtggca
     8761 tggttcgtac aaaccaaatg cggtgttctt gtctggcaaa gaggccaagg acgcagatgg
     8821 caacaaaccc aaggaacctg ggataacgga ggcttcatcg gagatgatat caccaaacat
     8881 gttgctggtg attataatac catttaggtg ggttgggttc ttaactagga tcatggcggc
     8941 agaatcaatc aattgatgtt gaaccttcaa tgtagggaat tcgttcttga tggtttcctc
     9001 cacagttttt ctccataatc ttgaagaggc caaaacatta gctttatcca aggaccaaat
     9061 aggcaatggt ggctcatgtt gtagggccat gaaagcggcc attcttgtga ttctttgcac
     9121 ttctggaacg gtgtattgtt cactatccca agcgacacca tcaccatcgt cttcctttct
     9181 cttaccaaag taaatacctc ccactaattc tctgacaaca acgaagtcag tacctttagc
     9241 aaattgtggc ttgattggag ataagtctaa aagagagtcg gatgcaaagt tacatggtct
     9301 taagttggcg tacaattgaa gttctttacg gatttttagt aaaccttgtt caggtctaac
     9361 actaccggta ccccatttag gaccacccac agcacctaac aaaacggcat cagccttctt
     9421 ggaggcttcc agcgcctcat ctggaagtgg aacacctgta gcatcgatag cagcaccacc
     9481 aattaaatga ttttcgaaat cgaacttgac attggaacga acatcagaaa tagctttaag
     9541 aaccttaatg gcttcggctg tgatttcttg accaacgtgg tcacctggca aaacgacgat
     9601 cttcttaggg gcagacatta gaatggtata tccttgaaat atatatatat attgctgaaa
     9661 tgtaaaaggt aagaaaagtt agaaagtaag acgattgcta accacctatt ggaaaaaaca
     9721 ataggtcctt aaataatatt gtcaacttca agtattgtga tgcaagcatt tagtcatgaa
     9781 cgcttctcta ttctatatga aaagccggtt ccggcgctct cacctttcct ttttctccca
     9841 atttttcagt tgaaaaaggt atatgcgtca ggcgacctct atgtgccacc aatcaacagt
     9901 taacgaacac gtggcggcgc ggaacgcctg gagccaggtc gcacgcttcc catatttact
     9961 tcgagcagtg taggtgtgac aaggtagcat gcagccttat gcctaacttg aaccgcagat
    10021 aaaggtctat acccgggatc cccactttct gagggttcag attcctgatg ctaccctaaa
    10081 attaccctgt tatccctaat aacttcgtat aatgtatgct atacgaagtt attccccagc
    10141 atgcctgcta ttctcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag
    10201 aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac
    10261 agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg
    10321 ctggcaacta gaaggcacag tcgaggctga tcagcgagct ctagttagaa ttcttagccc
    10381 tcccacacat aaccagaggg cagcaattca cgaatcccaa ctgccgtcgg ctgtccatca
    10441 ctgtccttca ctatggcttt gatcccagga tgcagatcga gaagcacctg tcggcaccgt
    10501 ccgcaggggc tcaagatgcc cctgttctca tttccgatcg cgacgataca agtcaggttg
    10561 ccagctgccg cagcagcagc agtgcccagc accacgagtt ctgcacaagg tcccccagta
    10621 aaatgatata cattgacacc agtgaagatg cggccgtcgc tagagagagc tgcgctggcg
    10681 acgctgtagt cctcagagat ggggatgctg ttgattgtag ccgttgctct ttcaatgagg
    10741 gtggattctt cttgagacaa aggcttggcc attgggccag gattctcctc gacgtcaccg
    10801 catgttagca gacttcctct gccctctcca ctgcccttgt acagctcgtc catgccgaga
    10861 gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg
    10921 tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag cacggggccg
    10981 tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg
    11041 ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc catgatatag
    11101 acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg
    11161 aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg
    11221 gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct
    11281 tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag
    11341 cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg cagcttgccg
    11401 gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg
    11461 gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc
    11521 ccggtgaaca gctcctcgcc cttgctcacc attggtaccc agcttctaga gatcacgaca
    11581 cctgaaatgg aagaaaaaaa ctttgaacca ctgtctgagg cttgagaatg aaccaagatc
    11641 caaactcaaa aagggcaaat tccaaggaga attacatcaa gtgccaagct ggcctaactt
    11701 cagtctccac ccactcagtg tggggaaact ccatcgcata aaacccctcc ccccaaccta
    11761 aagacgacgt actccaaaag ctcgagaact aatcgaggtg cctggacagc gcccggtact
    11821 ccgtggagtc acatgaagcg acggctgagg acggaaaggc ccttttcctt tgtgtgggtg
    11881 actcacccgc ccgctctccc gagcgccgcg tcctccattt tgagctccct gcagtagggc
    11941 cgggaagcgg ccatctttcc gctcacgcaa ctggtgccga ccgggccagc cttgccgccc
    12001 agggcggggc gatacacggc ggcgcgagac taggcaccag agcaggccgg ccagcttgag
    12061 actacccccg tccgattctc ggtggccgcg ctcgcaggcc ccgcctcgcc gaacatgtgc
    12121 gctgggacgc acgggccccg tcgccgcccg cggccccaaa aaccgaaata ccagtgtgca
    12181 gatcttggcc cgcatttaca agactatctt gccagaaaaa aagcgtcgca gcaggtcatc
    12241 aaaaatttta aatggctaga gacttatcga aagcagcgag acaggcgcga aggtgccacc
    12301 agattcgcac gcggcggccc cagcgcccag gccaggcctc aactcaagca cgaggcgaag
    12361 gggctcctta agcgcaaggc ctcgaactct cccacccact tccaacccga agctcgggat
    12421 caagaatcac gtactgcagc caggtggaag taattcaagg cacgcaaggg ccataacccg
    12481 taaagaggcc aggcccgcgg gaaccacaca cggcacttac ctgtgttctg gcggcaaacc
    12541 cgttgcgaaa aagaacgttc acggcgacta ctgcacttat atacggttct cccccaccct
    12601 cgggaaaaag gcggagccag tacacgacat cactttccca gtttaccccg cgccaccttc
    12661 tctaggcacc ggttcaattg ccgacccctc cccccaactt ctcggggact gtgggcgatg
    12721 tgcgctctgc ccactgacgg gcaccggagc cataacttcg tataaagtat cctatacgaa
    12781 gttatattac cctgttatcc ctaataatct ataaatgttg aggtttacag ttctcggggc
    12841 gagatcaaac ctaagtatat agtcctgtcc gaagccttgg ttcacatact cgccacgctt
    12901 gaaagcgcgt catgcttttc gctgactcgc tagctctatg cgtggcaatc taagtgggag
    12961 gtctgtcgca aggcttagaa actaaatatt ggtagctacc ttaggcccgt ccctgcaggt
    13021 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
    13081 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
    13141 tttagttagc ctcccccatc tcccgggcaa acgtgcgcgc caggtcgcat atcgtcggta
    13201 tggagccggg ggtggtgacg tgggtctgga ccatcccgga ggtaagttgc agcagggcgt
    13261 cccggcagcc ggcgggcgat tggtcgtaat ccaggataaa gacgtgcatg gaacggaggc
    13321 gtttggccaa gacgtccaag gcccaggcaa acacgttata caggtcgccg ttgggggcca
    13381 gcaactcggg ggcccgaaac agggtaaata acgtgtcccc gatatggggt cgtgggcccg
    13441 cgttgctctg gggctcggca ccctggggcg gcacggcggt ccccgaaagc tgtccccagt
    13501 cctcccgcca cgacccgccg cattgcagat accgcaccgt attggcaagt agcccgtaaa
    13561 cgcggcgaat cgcagccagc atagccaggt ccagccgctc gccggggcgc tggcgtttgg
    13621 ccaggcggtc gatgtgtctg tcctccggaa gggccccaag cacgatgttg gtgccgggca
    13681 aggtcggcgg gatgagggcc acgaacgcca gcacggcctg gggggtcatg ctgcccataa
    13741 ggtagcgcgc ggcggggtag cacaggaggg cggcgatggg atggcggtcg aagatgaggg
    13801 tgagggccgg gggcggggca tgtgagctcc cagcctcccc cccgatatga ggagccagaa
    13861 cggcgtcggt cacggcataa ggcatcccca ttgttatctg ggcgcttgtc attaccaccg
    13921 ccgcgtcccc ggcggatatc tcaccctggt cgaggcggtg ttgtgtggtg tagatgttcg
    13981 cgattgtctc ggaagccccc agcacccgcc agtaagtcat cggctcgggt acgtagacga
    14041 tatcgtcgcg cgaacccagg gccaccagca gttgcgtggt ggtggttttc cccatcccgt
    14101 ggggaccgtc tatataaacc cgcagtagcg tgggcatcct ccaacgaggg agccgactgc
    14161 cgacgtgcgc tccggaggct tgcagaatgc ggaacaccgc gcgggcagga acagggccca
    14221 cactaccgcc ccacaccccg cctcccgcac cgccccttcc cagccgctgc tctcggcgcg
    14281 ccctgctgag cagccgctat tggccacagc ccatcgcggt cggcgcgctg ccattgctcc
    14341 ctggcgctgt ccgtctgcga gggtactagt cagacgtgcg gcttccgttt gtcacgtccg
    14401 gcacgccgcg aaccgcaagg aaccttcccg acttaggggc ggagcaggaa gcgtcgccgg
    14461 ggggcccaca agggtagcgg cgaagatccg ggtgacgctg cgaacggacg tgaagaatgt
    14521 gcgaggccca gggtcggcgc cgctgcgttt cccggaacca cgcccagagc agccgcgtcc
    14581 ctgcgcaaac ccagggctgc cttggaaaag gcgcaacccc aacccccctg caggcgtctc
    14641 tgagacataa ccgtgcgcaa aatgttgtgg ataagcagga cacagcagca atccaccgca
    14701 ggcatacaac cgcacaccga ggttactccg ttctacaggt tacgacgaca tgtcaatact
    14761 tgcccttgac aggcattgat ggaatcgtag tctcacgctg atagtctgat cgacaataca
    14821 agtgggaccg t
//
