LOCUS       sequence-365374-       13328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Big-IN positive control payload encoding EF1a-driven GFP-T2A-BSD..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13328)
  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 13328)
  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
FEATURES             Location/Qualifiers
     source          1..13328
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(183..216)
                     /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)."
     promoter        217..1395
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          448..1386
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1343..1363
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             join(1416..1418,1419..1421,1422..2132)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1416..1418,1419..1421,1422..2132)
                     /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     complement(1461..1482)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1722..1741)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2069..2090
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2142..2195
                     /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             2196..2594
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(2622..2639)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2628..2835
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2836..2869)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3350..4444
                     /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"
     CDS             complement(5021..5836)
                     /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             complement(5021..5836)
                     /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     5744..5763
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5837..5939)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     mobile_element  5967..6734
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     misc_feature    6736..7239
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     rep_origin      8090..8704
                     /label=oriV
                     /note="origin of replication for the bacterial F plasmid"
     rep_origin      8181..8712
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      8780..8999
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             9090..9845
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     misc_feature    9848..10098
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             10424..11599
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     CDS             11599..12570
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     misc_feature    12643..13116
                     /gene="sopC"
                     /label=sopC
                     /note="centromere-like partitioning region of the bacterial
                     F plasmid"
ORIGIN
        1 gacagacctc ccacttagat tgccacgcat agagctagcg agtcagcgaa aagcatgacg
       61 cgctttcaag cgtggcgagt atgtgaacca aggcttcgga caggactata tacttaggtt
      121 tgatctcgcc ccgagaactg taaacctcaa catttataga ttattaggga taacagggta
      181 atataacttc gtataggata ctttatacga agttatggct ccggtgcccg tcagtgggca
      241 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt
      301 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
      361 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
      421 cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc
      481 ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg
      541 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa
      601 ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg
      661 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa
      721 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc
      781 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg
      841 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg
      901 gggtagtctc aagctggccg gcctgctctg gtgcctagtc tcgcgccgcc gtgtatcgcc
      961 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg
     1021 cttcccggcc ctactgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg
     1081 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac
     1141 tccacggagt accgggcgct gtccaggcac ctcgattagt tctcgagctt ttggagtacg
     1201 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg
     1261 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt
     1321 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca
     1381 tttcaggtgt cgtgatctct agaagctggg taccaatggt gagcaagggc gaggagctgt
     1441 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1501 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     1561 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     1621 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1681 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     1741 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1801 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     1861 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     1921 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1981 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     2041 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     2101 ggatcactct cggcatggac gagctgtaca agggcagtgg agagggcaga ggaagtctgc
     2161 taacatgcgg tgacgtcgag gagaatcctg gcccaatggc caagcctttg tctcaagaag
     2221 aatccaccct cattgaaaga gcaacggcta caatcaacag catccccatc tctgaggact
     2281 acagcgtcgc cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat
     2341 atcattttac tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg
     2401 cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc
     2461 cctgcggacg gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga
     2521 aggacagtga tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg
     2581 tgtgggaggg ctaagaattc taactagagc tcgctgatca gcctcgactg tgccttctag
     2641 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2701 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2761 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag
     2821 caggcatgct ggggaataac ttcgtatagc atacattata cgaagttatt agggataaca
     2881 gggtaatttt agggtagcat caggaatctg aaccctcaga aagtggggat cccgggtata
     2941 gacctttatc tgcggttcaa gttaggcata aggctgcatg ctaccttgtc acacctacac
     3001 tgctcgaagt aaatatggga agcgtgcgac ctggctccag gcgttccgcg ccgccacgtg
     3061 ttcgttaact gttgattggt ggcacataga ggtcgcctga cgcatatacc tttttcaact
     3121 gaaaaattgg gagaaaaagg aaaggtgaga gcgccggaac cggcttttca tatagaatag
     3181 agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat attatttaag
     3241 gacctattgt tttttccaat aggtggttag caatcgtctt actttctaac ttttcttacc
     3301 ttttacattt cagcaatata tatatatatt tcaaggatat accattctaa tgtctgcccc
     3361 taagaagatc gtcgttttgc caggtgacca cgttggtcaa gaaatcacag ccgaagccat
     3421 taaggttctt aaagctattt ctgatgttcg ttccaatgtc aagttcgatt tcgaaaatca
     3481 tttaattggt ggtgctgcta tcgatgctac aggtgttcca cttccagatg aggcgctgga
     3541 agcctccaag aaggctgatg ccgttttgtt aggtgctgtg ggtggtccta aatggggtac
     3601 cggtagtgtt agacctgaac aaggtttact aaaaatccgt aaagaacttc aattgtacgc
     3661 caacttaaga ccatgtaact ttgcatccga ctctctttta gacttatctc caatcaagcc
     3721 acaatttgct aaaggtactg acttcgttgt tgtcagagaa ttagtgggag gtatttactt
     3781 tggtaagaga aaggaagacg atggtgatgg tgtcgcttgg gatagtgaac aatacaccgt
     3841 tccagaagtg caaagaatca caagaatggc cgctttcatg gccctacaac atgagccacc
     3901 attgcctatt tggtccttgg ataaagctaa tgttttggcc tcttcaagat tatggagaaa
     3961 aactgtggag gaaaccatca agaacgaatt ccctacattg aaggttcaac atcaattgat
     4021 tgattctgcc gccatgatcc tagttaagaa cccaacccac ctaaatggta ttataatcac
     4081 cagcaacatg tttggtgata tcatctccga tgaagcctcc gttatcccag gttccttggg
     4141 tttgttgcca tctgcgtcct tggcctcttt gccagacaag aacaccgcat ttggtttgta
     4201 cgaaccatgc cacggttctg ctccagattt gccaaagaat aaggtcaacc ctatcgccac
     4261 tatcttgtct gctgcaatga tgttgaaatt gtcattgaac ttgcctgaag aaggtaaggc
     4321 cattgaagat gcagttaaaa aggttttgga tgcaggtatc agaactggtg atttaggtgg
     4381 ttccaacagt accaccgaag tcggtgatgc tgtcgccgaa gaagttaaga aaatccttgc
     4441 ttaaaaagat tctctttttt tatgatattt gtacataaac tttataaatg aaattcataa
     4501 tagaaacgac acgaaattac aaaatggaat atgttcatag ggtagacgaa actatatacg
     4561 caatctacat acatttatca agaaggagaa aaaggaggat gtaaaggaat acaggtaagc
     4621 aaattgatac taatggctca acgtgataag gaaaaagaat tgcactttaa cattaatatt
     4681 gacaaggagg agggcaccgc ggccgcccaa ctacgcagcc gatcaacaac agaaacgcga
     4741 cggtcgagat gaggaacgca cactggagcc tttatcagat ggtgttgatt tcaattactc
     4801 cggagcgtga tgcggatcaa agtaaccgcg atggaaggtg ggggactcga aatagatccg
     4861 ttcttagccc tgcgaaacat ctactgtaca ccacatgcaa cagccacaat tctcatgttt
     4921 gacagcttat catcgaattt ctgccattca tccgcttatt atcacttatt caggcgtagc
     4981 aaccaggcgt ttaagggcac caataactgc cttaaaaaaa ttagaaaaac tcatcgagca
     5041 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     5101 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     5161 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     5221 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     5281 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     5341 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcc agacgaaata
     5401 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     5461 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     5521 ctgtttttcc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     5581 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     5641 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     5701 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     5761 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc
     5821 gttgaatatg gctcatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata
     5881 cgtccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa
     5941 cgtccctggg tccttttcat cacgtgggtg atgctgccaa cttactgatt tagtgtatga
     6001 tggtgttttt gaggtgctcc agtggcttct gtttctatca gctgtccctc ctgttcagct
     6061 actgacgggg tggtgcgtaa cggcaaaagc accgccggac atcagcgcta tctctgctct
     6121 cactgccgta aaacatggca actgcagttc acttacaccg cttctcaacc cggtacgcac
     6181 cagaaaatca ttgatatggc catgaatggc gttggatgcc gggcaacagc ccgcattatg
     6241 ggcgttggcc tcaacacgat tttacgtcac ttaaaaaact caggccgcag tcggtaacct
     6301 cgcgcataca gccgggcagt gacgtcatcg tctgcgcgga aatggacgaa cagtggggct
     6361 atgtcggggc taaatcgcgc cagcgctggc tgttttacgc gtatgacagt ctccggaaga
     6421 cggttgttgc gcacgtattc ggtgaacgca ctatggcgac gctggggcgt cttatgagcc
     6481 tgctgtcacc ctttgacgtg gtgatatgga tgacggatgg ctggccgctg tatgaatccc
     6541 gcctgaaggg aaagctgcac gtaatcagca agcgatatac gcagcgaatt gagcggcata
     6601 acctgaatct gaggcagcac ctggcacggc tgggacggaa gtcgctgtcg ttctcaaaat
     6661 cggtggagct gcatgacaaa gtcatcgggc attatctgaa cataaaacac tatcaataag
     6721 ttggagtcat tacccatcac gtgctataaa aataattata atttaaattt tttaatataa
     6781 atatataaat taaaaataga aagtaaaaaa agaaattaaa gaaaaaatag tttttgtttt
     6841 ccgaagatgt aaaagactct agggggatcg ccaacaaata ctacctttta tcttgctctt
     6901 cctgctctca ggtattaatg ccgaattgtt tcatcttgtc tgtgtagaag accacacacg
     6961 aaaatcctgt gattttacat tttacttatc gttaatcgaa tgtatatcta tttaatctgc
     7021 ttttcttgtc taataaatat atatgtaaag tacgcttttt gttgaaattt tttaaacctt
     7081 tgtttatttt tttttcttca ttccgtaact cttctacctt ctttatttac tttctaaaat
     7141 ccaaatacaa aacataaaaa taaataaaca cagagtaaat tcccaaatta ttccatcatt
     7201 aaaagatacg aggcgcgtgt aagttacagg caagcgatct tttcgccaaa agttggccca
     7261 gggcttcccg gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt
     7321 cacaggtatt tattcgcgat aagctcatgg agcggcgtaa ccgtcgcaca ggaaggacag
     7381 agaaagcgcg gatctgggaa gtgacggaca gaacggtcag gacctggatt ggggaggcgg
     7441 ttgccgccgc tgctgctgac ggtgtgacgt tctctgttcc ggtcacacca catacgttcc
     7501 gccattccta tgcgatgcac atgctgtatg ccggtatacc gctgaaagtt ctgcaaagcc
     7561 tgatgggaca taagtccatc agttcaacgg aagtctacac gaaggttttt gcgctggatg
     7621 tggctgcccg gcaccgggtg cagtttgcga tgccggagtc tgatgcggtt gcgatgctga
     7681 aacaattatc ctgagaataa atgccttggc ctttatatgg aaatgtggaa ctgagtggat
     7741 atgctgtttt tgtctgttaa acagagaagc tggctgttat ccactgagaa gcgagcgaaa
     7801 cagtcgggaa aatctcccat tatcgtagag atccgcatta ttaatctcag gagcctgtgt
     7861 agcgtttata ggaagtagtg ttctgtcatg atgcctgcaa gcggtaacga aaacgatttg
     7921 aatatgcctt caggaacaat agaaatcttc gtgcggtgtt acgttgaagt ggagcggatt
     7981 atgtcagcaa tggacagaac aacctaatga acacagaacc atgatgtggt ctgtcctttt
     8041 acagccagta gtgctcgccg cagtcgagcg acagggcgaa gccctcgagc tggttgccct
     8101 cgccgctggg ctggcggctg tctatggccc tgcaaacgcg ccagaaacgc cgtcgaagcc
     8161 gtgtgcgaga caccgcggcc ggccgccggc gttgtggata cctcgcggaa aacttggccc
     8221 tcactgacag atgaggggcg gacgttgaca cttgaggggc cgactcaccc ggcgcggcgt
     8281 tgacagatga ggggcaggct cgatttcggc cggcgacgtg gagctggaca gcctcgcaaa
     8341 tcggcgaaaa cgcctgattt tacgcgagtt tcccacagat gatgtggaca agcctgggga
     8401 taagtgccct gcggtattga cacttgaggg gcgcgactac tgacagatga ggggcgcgat
     8461 ccttgacact tgaggggcag agtgctgaca gatgaggggc gcacctattg acatttgagg
     8521 ggctgtccac aggcagaaaa tccagcattt gcaagggttt ccgcccgttt ttcggccacc
     8581 gctaacctgt cttttaacct gcttttaaac caatatttat aaaccttgtt tttaaccagg
     8641 gctgcgccct gtgcgcgtga ccgcgcacgc cgaagggggg tgccccccct tctcgaaccc
     8701 tcccggtcga gtgagcgagg aagcaccagg gaacagcact tatatattct gcttacacac
     8761 gatgcctgaa aaaacttccc ttggggttat ccacttatcc acggggatat ttttataatt
     8821 atttttttta tagtttttag atcttctttt ttagagcgcc ttgtaggcct ttatccatgc
     8881 tggttctaga gaaggtgttg tgacaaattg ccctttcagt gtgacaaatc accctcaaat
     8941 gacagtcctg tctgtgacaa attgccctta accctgtgac aaattgccct cagaagaagc
     9001 tgttttttca caaagttatc cctgcttatt gactcttttt tatttagtgt gacaatctaa
     9061 aaacttgtca cacttcacat ggatctgtca tggcggaaac agcggttatc aatcacaaga
     9121 aacgtaaaaa tagcccgcga atcgtccagt caaacgacct cactgaggcg gcatatagtc
     9181 tctcccggga tcaaaaacgt atgctgtatc tgttcgttga ccagatcaga aaatctgatg
     9241 gcaccctaca ggaacatgac ggtatctgcg agatccatgt tgctaaatat gctgaaatat
     9301 tcggattgac ctctgcggaa gccagtaagg atatacggca ggcattgaag agtttcgcgg
     9361 ggaaggaagt ggttttttat cgccctgaag aggatgccgg cgatgaaaaa ggctatgaat
     9421 cttttccttg gtttatcaaa cgtgcgcaca gtccatccag agggctttac agtgtacata
     9481 tcaacccata tctcattccc ttctttatcg ggttacagaa ccggtttacg cagtttcggc
     9541 ttagtgaaac aaaagaaatc accaatccgt atgccatgcg tttatacgaa tccctgtgtc
     9601 agtatcgtaa gccggatggc tcaggcattg tctctctgaa aatcgactgg atcatagagc
     9661 gttaccagct gcctcaaagt taccagcgta tgcctgactt ccgccgccgc ttcctgcagg
     9721 tctgtgttaa tgagatcaac agcagaactc caatgcgcct ctcatacatt gagaaaaaga
     9781 aaggccgcca gacgactcat atcgtatttt ccttccgcga tatcacttcc atgacgacag
     9841 gatagtctga gggttatctg tcacagattt gagggtggtt cgtcacattt gttctgacct
     9901 actgagggta atttgtcaca gttttgctgt ttccttcagc ctgcatggat tttctcatac
     9961 tttttgaact gtaattttta aggaagccaa atttgagggc agtttgtcac agttgatttc
    10021 cttctctttc ccttcgtcat gtgacctgat atcgggggtt agttcgtcat cattgatgag
    10081 ggttgattat cacagtttat tactctgaat tggctatccg cgtgtgtacc tctacctgga
    10141 gtttttccca cggtggatat ttcttcttgc gctgagcgta agagctatct gacagaacag
    10201 ttcttctttg cttcctcgcc agttcgctcg ctatgctcgg ttacacggct gcggcgagcg
    10261 ctagtgataa taagtgactg aggtatgtgc tcttcttatc tccttttgta gtgttgctct
    10321 tattttaaac aactttgcgg ttttttgatg actttgcgat tttgttgttg ctttgcagta
    10381 aattgcaaga tttaataaaa aaacgcaaag caatgattaa aggatgttca gaatgaaact
    10441 catggaaaca cttaaccagt gcataaacgc tggtcatgaa atgacgaagg ctatcgccat
    10501 tgcacagttt aatgatgaca gcccggaagc gaggaaaata acccggcgct ggagaatagg
    10561 tgaagcagcg gatttagttg gggtttcttc tcaggctatc agagatgccg agaaagcagg
    10621 gcgactaccg cacccggata tggaaattcg aggacgggtt gagcaacgtg ttggttatac
    10681 aattgaacaa attaatcata tgcgtgatgt gtttggtacg cgattgcgac gtgctgaaga
    10741 cgtatttcca ccggtgatcg gggttgctgc ccataaaggt ggcgtttaca aaacctcagt
    10801 ttctgttcat cttgctcagg atctggctct gaaggggcta cgtgttttgc tcgtggaagg
    10861 taacgacccc cagggaacag cctcaatgta tcacggatgg gtaccagatc ttcatattca
    10921 tgcagaagac actctcctgc ctttctatct tggggaaaag gacgatgtca cttatgcaat
    10981 aaagcccact tgctggccgg ggcttgacat tattccttcc tgtctggctc tgcaccgtat
    11041 tgaaactgag ttaatgggca aatttgatga aggtaaactg cccaccgatc cacacctgat
    11101 gctccgactg gccattgaaa ctgttgctca tgactatgat gtcatagtta ttgacagcgc
    11161 gcctaacctg ggtatcggca cgattaatgt cgtatgtgct gctgatgtgc tgattgttcc
    11221 cacgcctgct gagttgtttg actacacctc cgcactgcag tttttcgata tgcttcgtga
    11281 tctgctcaag aacgttgatc ttaaagggtt cgagcctgat gtacgtattt tgcttaccaa
    11341 atacagcaat agcaatggct ctcagtcccc gtggatggag gagcaaattc gggatgcctg
    11401 gggaagcatg gttctaaaaa atgttgtacg tgaaacggat gaagttggta aaggtcagat
    11461 ccggatgaga actgtttttg aacaggccat tgatcaacgc tcttcaactg gtgcctggag
    11521 aaatgctctt tctatttggg aacctgtctg caatgaaatt ttcgatcgtc tgattaaacc
    11581 acgctgggag attagataat gaagcgtgcg cctgttattc caaaacatac gctcaatact
    11641 caaccggttg aagatacttc gttatcgaca ccagctgccc cgatggtgga ttcgttaatt
    11701 gcgcgcgtag gagtaatggc tcgcggtaat gccattactt tgcctgtatg tggtcgggat
    11761 gtgaagttta ctcttgaagt gctccggggt gatagtgttg agaagacctc tcgggtatgg
    11821 tcaggtaatg aacgtgacca ggagctgctt actgaggacg cactggatga tctcatccct
    11881 tcttttctac tgactggtca acagacaccg gcgttcggtc gaagagtatc tggtgtcata
    11941 gaaattgccg atgggagtcg ccgtcgtaaa gctgctgcac ttaccgaaag tgattatcgt
    12001 gttctggttg gcgagctgga tgatgagcag atggctgcat tatccagatt gggtaacgat
    12061 tatcgcccaa caagtgctta tgaacgtggt cagcgttatg caagccgatt gcagaatgaa
    12121 tttgctggaa atatttctgc gctggctgat gcggaaaata tttcacgtaa gattattacc
    12181 cgctgtatca acaccgccaa attgcctaaa tcagttgttg ctcttttttc tcaccccggt
    12241 gaactatctg cccggtcagg tgatgcactt caaaaagcct ttacagataa agaggaatta
    12301 cttaagcagc aggcatctaa ccttcatgag cagaaaaaag ctggggtgat atttgaagct
    12361 gaagaagtta tcactctttt aacttctgtg cttaaaacgt catctgcatc aagaactagt
    12421 ttaagctcac gacatcagtt tgctcctgga gcgacagtat tgtataaggg cgataaaatg
    12481 gtgcttaacc tggacaggtc ccgtgttcca actgagtgta tagagaaaat tgaggccatt
    12541 cttaaggaac ttgaaaagcc agcaccctga tgcgacctcg ttttagtcta cgtttatctg
    12601 tctttactta atgtcctttg ttacaggcca gaaagcataa ctggcctgaa tattctctct
    12661 gggcccactg ttccacttgt atcgtcggtc tgataatcag actgggacca cggtcccact
    12721 cgtatcgtcg gtctgattat tagtctggga ccacggtccc actcgtatcg tcggtctgat
    12781 tattagtctg ggaccacggt cccactcgta tcgtcggtct gataatcaga ctgggaccac
    12841 ggtcccactc gtatcgtcgg tctgattatt agtctgggac catggtccca ctcgtatcgt
    12901 cggtctgatt attagtctgg gaccacggtc ccactcgtat cgtcggtctg attattagtc
    12961 tggaaccacg gtcccactcg tatcgtcggt ctgattatta gtctgggacc acggtcccac
    13021 tcgtatcgtc ggtctgatta ttagtctggg accacgatcc cactcgtgtt gtcggtctga
    13081 ttatcggtct gggaccacgg tcccacttgt attgtcgatc agactatcag cgtgagacta
    13141 cgattccatc aatgcctgtc aagggcaagt attgacatgt cgtcgtaacc tgtagaacgg
    13201 agtaacctcg gtgtgcggtt gtatgcctgc ggtggattgc tgctgtgtcc tgcttatcca
    13261 caacattttg cgcacggtta tgtggacggg cctaaggtag ctaccaatat ttagtttcta
    13321 agccttgc
//
