LOCUS       sequence-489344-       12656 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-GT donor plasmid with downstream tandem syntax for all-in-one 
            doxycycline-inducible expression of Ngn2, ISL1 and LHX3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12656)
  AUTHORS   Blanch-Asensio A, Ploessl DS, Wang NB, Mummery CL, Galloway KE, 
            Davis RP
  TITLE     STRAIGHT-IN Dual: a platform for dual, single-copy integrations of 
            DNA payloads and gene circuits into human induced pluripotent stem 
            cells.
  JOURNAL   bioRxiv [Preprint]. 2024 Oct 17:2024.10.17.616637. doi: 
            10.1101/2024.10.17.616637.
  PUBMED    39464154
REFERENCE   2  (bases 1 to 12656)
  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: "bioRxiv 
            [Preprint]. 2024 Oct 17:2024.10.17.616637. doi: 
            10.1101/2024.10.17.616637."
COMMENT     Sequence Label: Bxb1-GT-Dox-AIO-TetOn_Ngn2-ISL1-LHX3_Down-Tandem
FEATURES             Location/Qualifiers
     source          1..12656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        253..463
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        275..471
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      322..457
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        558..1736
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          789..1727
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1684..1704
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1744..1753
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1756..1793
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    complement(1802..1835)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1881..1899
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1917..1935
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1953..1971
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1989..2007
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2025..2043
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2061..2079
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2097..2115
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2160..2184
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             3097..3153
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     regulatory      3944..3953
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4225..4278
                     /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"
     misc_feature    5494..6082
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5547..5567)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5965..5976)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     enhancer        6139..6518
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6520..6795
                     /label=chicken beta-actin promoter
     primer_bind     7688..7710
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     7772..7791
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             7823..8566
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             8567..8620
                     /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             8621..9328
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     CDS             9353..9373
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9377..9397
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9401..9421
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(9486..9503)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9492..9716
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(9745..9777)
                     /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)."
     primer_bind     complement(9785..9802)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9785..9801)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9794..9816)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10014..10469
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10101..10120)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10311..10332
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10486..10505)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     10605..10627
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(10665..10683)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        10751..10855
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10856..10924,10925..11716)
                     /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(11074..11093)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11887..12475
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12376..12395
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12629..12646
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctta
      241 attccctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
      301 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
      361 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
      421 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctttgc
      481 aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct
      541 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
      601 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
      661 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
      721 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
      781 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc
      841 cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc
      901 gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg
      961 cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc
     1021 gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg
     1081 cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta
     1141 tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg
     1201 cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc
     1261 ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc
     1321 tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag
     1381 ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa
     1441 ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc
     1501 cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg
     1561 aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag
     1621 cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca
     1681 ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaggaa
     1741 ttcgccacca tggccggctt gtcgacgacg gcggtctccg tcgtcaggat catccggatc
     1801 cataacttcg tatagcatac attatacgaa gttatcatga tattcggcaa gcaggcatcg
     1861 actagttaat taaggtttac tccctatcag tgatagagaa cgtatgaaga gtttactccc
     1921 tatcagtgat agagaacgta tgcagacttt actccctatc agtgatagag aacgtataag
     1981 gagtttactc cctatcagtg atagagaacg tatgaccagt ttactcccta tcagtgatag
     2041 agaacgtatc tacagtttac tccctatcag tgatagagaa cgtatatcca gtttactccc
     2101 tatcagtgat agagaacgta taagctttag gcgtgtacgg tgggcgccta taaaagcaga
     2161 gctcgtttag tgaaccgtca gatcgcctgg agcaattcca caacactttt gtcttatacc
     2221 aactttccgt accacttcct accctcgtaa acttaagaat tcgccaccat gttcgtcaaa
     2281 tccgagacct tggagttgaa ggaggaagag gacgtgttag tgctgctcgg atcggcctcc
     2341 cccgccttgg cggccctgac cccgctgtca tccagcgccg acgaagaaga ggaggaggag
     2401 ccgggcgcgt caggcggggc gcgtcggcag cgcggggctg aggccgggca gggggcgcgg
     2461 ggcggcgtgg ctgcgggtgc ggagggctgc cggcccgcac ggctgctggg tctggtacac
     2521 gattgcaaac ggcgcccttc ccgggcgcgg gccgtctccc gaggcgccaa gacggccgag
     2581 acggtgcagc gcatcaagaa gacccgtaga ctgaaggcca acaaccgcga gcgaaaccgc
     2641 atgcacaacc tcaacgcggc actggacgcg ctgcgcgagg tgctccccac gttccccgag
     2701 gacgccaagc tcaccaagat cgagaccctg cgcttcgccc acaactacat ctgggcactc
     2761 accgagaccc tgcgcctggc ggatcactgc gggggcggcg gcgggggcct gccgggggcg
     2821 ctcttctccg aggcagtgtt gctgagcccg ggaggcgcca gcgccgccct gagcagcagc
     2881 ggagacagcc cctcgcccgc ctccacgtgg agttgcacca acagccccgc gccgtcctcc
     2941 tccgtgtcct ccaattccac ctccccctac agctgcactt tatcgcccgc cagcccggcc
     3001 gggtcagaca tggactattg gcagccccca cctcccgaca agcaccgcta tgcacctcac
     3061 ctccccatag ccagggattg tatcttggga tccggagcta ctaacttcag cctgctgaag
     3121 caggctggag acgtggagga gaaccctgga cctatgggag acatgggaga tccaccaaaa
     3181 aaaaaacgtc tgatttccct atgtgttggt tgcggcaatc agattcacga tcagtatatt
     3241 ctgagggttt ctccggattt ggaatggcat gcggcatgtt tgaaatgtgc ggagtgtaat
     3301 cagtatttgg acgagagctg tacatgcttt gttagggatg ggaaaaccta ctgtaaaaga
     3361 gattatatca ggttgtacgg gatcaaatgc gccaagtgca gcatcggctt cagcaagaac
     3421 gacttcgtga tgcgtgcccg ctccaaggtg tatcacatcg agtgtttccg ctgtgtggcc
     3481 tgcagccgcc agctcatccc tggggacgaa tttgcgcttc gggaggacgg tctcttctgc
     3541 cgagcagacc acgatgtggt ggagagggcc agtctaggcg ctggcgaccc gctcagtccc
     3601 ctgcatccag cgcggccact gcaaatggca gcggagccca tctccgccag gcagccggcc
     3661 ctgcggcccc acgtccacaa gcagccggag aagaccaccc gcgtgcggac tgtgctgaac
     3721 gagaagcagc tgcacacctt gcggacctgc tacgccgcaa acccgcggcc agatgcgctc
     3781 atgaaggagc aactggtaga gatgacgggc ctcagtcccc gtgtgatccg ggtctggttt
     3841 caaaacaagc ggtgcaagga caagaagcga agcatcatga tgaagcaact ccagcagcag
     3901 cagcccaatg acaaaactaa tatccagggg atgacaggaa ctcccatggt ggctgccagt
     3961 ccagagagac acgacggtgg cttacaggct aacccagtgg aagtacaaag ttaccagcca
     4021 ccttggaaag tactgagcga cttcgccttg cagagtgaca tagatcagcc tgcttttcag
     4081 caactggtca atttttcaga aggaggaccg ggctctaatt ccactggcag tgaagtagca
     4141 tcaatgtcct ctcaacttcc agatacacct aacagcatgg tagccagtcc tattgaggca
     4201 ttgatgcaca gcagaggctc tggcgagggc agaggcagcc tgctgacatg tggcgacgtg
     4261 gaagagaacc ctggccccgg taccatggaa gccagaggcg aactgggccc tgccagagaa
     4321 tctgccggcg gagatctgct gctggccctg ctggctagaa gggccgacct gagaagagaa
     4381 atccccctgt gcgccggctg cgaccagcac atcctggacc ggtttatcct gaaggccctg
     4441 gacagacact ggcacagcaa gtgcctgaag tgcagcgact gccacacccc cctggccgag
     4501 agatgcttta gcagaggcga gagcgtgtac tgcaaggacg acttcttcaa gagattcggc
     4561 accaagtgcg ccgcctgcca gctgggaatt cctcctacac aggtcgtgcg gagagcccag
     4621 gacttcgtgt accatctgca ctgcttcgcc tgcgtcgtgt gcaagagaca gctggccacc
     4681 ggcgacgagt tctacctgat ggaagatagc cggctcgtgt gtaaagccga ctacgagaca
     4741 gccaagcaga gagaggccga ggccaccgcc aaaagaccca gaaccaccat caccgccaag
     4801 cagctggaaa ccctgaagtc cgcctacaac accagcccca agcccgccag acatgtgcgg
     4861 gaacagctga gcagcgagac aggcctggac atgagagtgg tgcaagtgtg gttccagaac
     4921 agacgggcca aagagaagcg gctgaagaag gacgccggca ggcagagatg gggccagtac
     4981 ttccggaaca tgaagcggtc cagaggcggc agcaagagcg acaaggacag cgtgcaggaa
     5041 ggccaggaca gcgacgccga ggtgtccttc ccagatgagc ctagcctggc cgaaatgggc
     5101 ccagccaatg gcctgtatgg cagcctggga gagccaaccc aggctctggg cagaccttct
     5161 ggcgccctgg gcaacttctc tctggaacac ggcggactgg ccggacccga gcagtataga
     5221 gaactgaggc ccggcagccc ttacggcgtg ccaccttctc cagctgcccc tcagtctctg
     5281 ccaggacctc agcctctgct gtccagcctg gtgtaccccg atacaagcct gggcctggtg
     5341 ccatctggcg ctccaggcgg acctcctcca atgagagtgc tggccggcaa tggccctagc
     5401 agcgatctga gcacaggcag ctctggcggc taccccgatt ttcctgccag ccctgcctcc
     5461 tggctggacg aagtggatca cgcccagttc tgaaatcaac ctctggatta caaaatttgt
     5521 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     5581 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     5641 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     5701 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     5761 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     5821 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     5881 tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc
     5941 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     6001 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     6061 tccctttggg ccgcctcccc gccctttggg ccgcctcccc gcgctagtgc tggggatgcg
     6121 gtgggctcta tgggtctcga cattgattat tgactagtta ttaatagtaa tcaattacgg
     6181 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     6241 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     6301 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     6361 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     6421 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     6481 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct
     6541 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt
     6601 aattattttg tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg
     6661 cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc
     6721 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
     6781 agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc
     6841 gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg
     6901 gacggccctt ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt
     6961 ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc
     7021 tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc
     7081 ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag
     7141 gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc
     7201 tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg
     7261 caaccccccc tgcacccccc tccccgagtt gctgagcgcg gcccggcttc gggtgcgggg
     7321 ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg
     7381 gtgccgggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc
     7441 cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag
     7501 ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc
     7561 gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt
     7621 ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc
     7681 agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt
     7741 catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat
     7801 cattttggca aagaatgtcg acatgagccg gctggacaag agcaaagtga tcaacagcgc
     7861 cctggaactg ctgaacggcg tgggcatcga gggcctgacc acccggaagc tggcccagaa
     7921 actgggcgtg gaacagccca ccctgtactg gcacgtgaag aacaagcggg ccctgctgga
     7981 cgccctgccc atcgagatgc tggaccggca ccacacccac agctgccctc tggaaggcga
     8041 gagctggcag gacttcctgc ggaacaacgc caagagctac agatgcgccc tgctgagcca
     8101 ccgggacggc gccaaagtgc acctgggcac cagacccacc gagaagcagt acgagacact
     8161 ggaaaaccag ctggccttcc tgtgccagca gggcttcagc ctggaaaacg ccctgtacgc
     8221 cctgagcgcc gtgggccact tcaccctggg ctgcgtgctg gaagaacagg aacaccaggt
     8281 cgccaaagag gaaagagaga cacccaccac cgacagcatg ccccccctgc tgaagcaggc
     8341 catcgagctg ttcgacagac agggcgccga gcccgccttc ctgttcggcc tggaactgat
     8401 catctgcggc ctcgagaagc agctgaagtg cgagagcggc ggacccaccg acgccctgga
     8461 cgacttcgac ctggacatgc tgcccgccga tgccctggat gattttgatc tggatatgct
     8521 ccctgctgac gctctcgatg acttcgatct cgatatgctg ccaggcgagg gcaggggaag
     8581 tcttctaaca tgcggggacg tggaggaaaa tcccggcccc gtgtctaagg gcgaagagct
     8641 gattaaggag aacatgcaca tgaagctgta catggagggc accgtggaca accatcactt
     8701 caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa
     8761 ggtggtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacta gcttcctcta
     8821 cggcagcaag accttcatca accacaccca gggcatcccc gacttcttca agcagtcctt
     8881 ccctgagggc ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc
     8941 tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt
     9001 gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggccttcac
     9061 cgagacgctg taccccgctg acggcggcct ggaaggcaga aacgacatgg ccctgaagct
     9121 cgtgggcggg agccatctga tcgcaaacgc caagaccaca tatagatcca agaaacccgc
     9181 taagaacctc aagatgcctg gcgtctacta tgtggactac agactggaaa gaatcaagga
     9241 ggccaacaac gagacctacg tcgagcagca cgaggtggca gtggccagat actgcgacct
     9301 ccctagcaaa ctggggcaca agcttaattc cggactcaga tctcgagctg atccaaaaaa
     9361 gaagagaaag gtagatccaa aaaagaagag aaaggtagat ccaaaaaaga agagaaaggt
     9421 aggatccacc ggatctagat aactgatcat aatcagccat accacatttg aaacccgctg
     9481 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     9541 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     9601 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     9661 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggctag
     9721 ccgctgcaag aactcttcct cacgataact tcgtatagga gactttatac gaagttaagc
     9781 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     9841 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     9901 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc
     9961 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
    10021 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
    10081 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
    10141 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
    10201 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
    10261 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
    10321 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
    10381 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
    10441 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
    10501 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
    10561 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
    10621 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
    10681 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
    10741 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
    10801 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
    10861 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
    10921 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
    10981 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
    11041 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
    11101 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
    11161 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
    11221 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
    11281 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
    11341 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
    11401 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
    11461 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
    11521 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
    11581 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
    11641 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
    11701 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
    11761 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
    11821 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
    11881 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
    11941 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
    12001 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
    12061 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
    12121 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
    12181 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
    12241 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
    12301 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
    12361 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
    12421 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
    12481 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
    12541 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
    12601 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaag
//
