LOCUS       sequence-234596-       11584 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid targeting human AAVS1 safe harbor locus to generate 
            GCaMP6s gene knockin. Contains two homologous arms flanking T2A-Puro
            resistance gene and CAG-driven GCaMP6s gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11584)
  AUTHORS   Jiang Y, Zhou Y, Bao X, Chen C, Randolph LN, Du J, Lian XL
  TITLE     An Ultrasensitive Calcium Reporter System via CRISPR-Cas9-Mediated 
            Genome Editing in Human Pluripotent Stem Cells.
  JOURNAL   iScience. 2018 Oct 12;9:27-35. doi: 10.1016/j.isci.2018.10.007.
  PUBMED    30368079
REFERENCE   2  (bases 1 to 11584)
  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: "iScience.";
            date: "2018-10-12"; volume: "9:27-35. doi"; pages: " 
            10.1016/j.isci.2018.10.007"
COMMENT     Sequence Label: AAVS1-Puro-CAG-GCaMP6s
FEATURES             Location/Qualifiers
     source          1..11584
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    37..840
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    847..872
                     /label=SA
                     /note="splice acceptor site"
     CDS             896..949
                     /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             959..1558
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(959..978)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1455..1475
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1590..1607)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1596..1820
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        1826..2205
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2207..2484
                     /label=chicken beta-actin promoter
     intron          2485..3501
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3425..3447
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3509..3528
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(3562..3570,3571..3588,3589..3591,3592..3624,3625..3627,
                     3628..3651,3652..3678,3679..3735,3736..3741,3742..4011,
                     4012..4029,4030..4464,4465..4470,4471..4914)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      3658..3667
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3948..3969
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4075..4096)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4336..4355)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    4945..5533
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4998..5018)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5416..5427)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5565..6041
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5764..5783)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(6151..6170)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6216..6271
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6270..6289)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6632..6648)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6656..6672
                     /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)."
     promoter        complement(join(6680..6686,6687..6704,6705..6710))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6725..6746
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    6820..7656
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(7690..7709)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7691..7709)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7716..7733)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7716..7732)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7725..7747)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             7870..8172
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     8051..8070
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(8575..8594)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     9189..9208
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(9569..10360,10361..10429))
                     /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     10192..10211
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10553..11141
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11042..11061
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11295..11312
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11429..11450
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11465..11470,11471..11488,11489..11495)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11503..11519
                     /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     11527..11543
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        11564..11582
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ctagctagtc ctgcaggttt aaacgaattc gccctttgct ttctctgacc agcattctct
       61 cccctgggcc tgtgccgctt tctgtctgca gcttgtggcc tgggtcacct ctacggctgg
      121 cccagatcct tccctgccgc ctccttcagg ttccgtcttc ctccactccc tcttcccctt
      181 gctctctgct gtgttgctgc ccaaggatgc tctttccgga gcacttcctt ctcggcgctg
      241 caccacgtga tgtcctctga gcggatcctc cccgtgtctg ggtcctctcc gggcatctct
      301 cctccctcac ccaaccccat gccgtcttca ctcgctgggt tcccttttcc ttctccttct
      361 ggggcctgtg ccatctctcg tttcttagga tggccttctc cgacggatgt ctcccttgcg
      421 tcccgcctcc ccttcttgta ggcctgcatc atcaccgttt ttctggacaa ccccaaagta
      481 ccccgtctcc ctggctttag ccacctctcc atcctcttgc tttctttgcc tggacacccc
      541 gttctcctgt ggattcgggt cacctctcac tcctttcatt tgggcagctc ccctaccccc
      601 cttacctctc tagtctgtgc tagctcttcc agccccctgt catggcatct tccaggggtc
      661 cgagagctca gctagtcttc ttcctccaac ccgggcccct atgtccactt caggacagca
      721 tgtttgctgc ctccagggat cctgtgtccc cgagctggga ccaccttata ttcccagggc
      781 cggttaatgt ggctctggtt ctgggtactt ttatctgtcc cctccacccc acagtggggc
      841 aagcttctga cctcttctct tcctcccaca gggcctcgag agatctggca gcggagaggg
      901 cagaggaagt cttctaacat gcggtgacgt ggaggagaat cccggcccta ggctcgagat
      961 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     1021 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     1081 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     1141 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     1201 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     1261 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     1321 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     1381 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     1441 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     1501 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgatc
     1561 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     1621 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1681 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     1741 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     1801 ggatgcggtg ggctctatgg gtctcgacat tgattattga ctagttatta atagtaatca
     1861 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     1921 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     1981 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     2041 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     2101 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     2161 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc
     2221 acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt
     2281 attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggggcg cgcgccaggc
     2341 ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat
     2401 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat
     2461 aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc
     2521 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg
     2581 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt
     2641 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg
     2701 gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc
     2761 gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt
     2821 gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg
     2881 aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg
     2941 gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg
     3001 ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg
     3061 caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg
     3121 cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt
     3181 ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg
     3241 aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc
     3301 ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc
     3361 cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg
     3421 gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat
     3481 gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat
     3541 tttggcaaag aattcgtcga catgggttct catcatcatc atcatcatgg tatggctagc
     3601 atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatctcgcc
     3661 accatggtcg actcatcacg tcgtaagtgg aataagacag gtcacgcagt cagagctata
     3721 ggtcggctga gctcactcga gaacgtctat atcaaggccg acaagcagaa gaacggcatc
     3781 aaggcgaact tccacatccg ccacaacatc gaggacggcg gcgtgcagct cgcctaccac
     3841 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     3901 agcgtgcagt ccaaactttc gaaagacccc aacgagaagc gcgatcacat ggtcctgctg
     3961 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggcggtacc
     4021 ggagggagca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     4081 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg tgagggcgat
     4141 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     4201 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     4261 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacat ccaggagcgc
     4321 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     4381 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     4441 ctggggcaca agctggagta caacctgccg gaccaactga ctgaagagca gatcgcagaa
     4501 tttaaagagg ctttctccct atttgacaag gacggggatg ggacaataac aaccaaggag
     4561 ctggggacgg tgatgcggtc tctggggcag aaccccacag aagcagagct gcaggacatg
     4621 atcaatgaag tagatgccga cggtgacggc acaatcgact tccctgagtt cctgacaatg
     4681 atggcaagaa aaatgaaata cagggacacg gaagaagaaa ttagagaagc gttcggtgtg
     4741 tttgataagg atggcaatgg ctacatcagt gcagcagagc ttcgccacgt gatgacaaac
     4801 cttggagaga agttaacaga tgaagaggtt gatgaaatga tcagggaagc agacatcgat
     4861 ggggatggtc aggtaaacta cgaagagttt gtacaaatga tgacagcgaa gtaaacgcgt
     4921 gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     4981 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     5041 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     5101 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     5161 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     5221 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     5281 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     5341 tcatcgtcct ttccttggct gctcgcctat gttgccacct ggattctgcg cgggacgtcc
     5401 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     5461 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     5521 gccgcctccc cgcatcgata ccgagcgctg ctcgagagat ctacgggtgg catccctgtg
     5581 acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg
     5641 tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg
     5701 ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg
     5761 gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg
     5821 cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat
     5881 gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg
     5941 ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc
     6001 tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgattttg taggtaacca
     6061 ggcgcgtgaa ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc
     6121 caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga
     6181 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc
     6241 aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca
     6301 tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc
     6361 tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt
     6421 ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt
     6481 tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac tagccagatt
     6541 tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct
     6601 cgacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     6661 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     6721 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     6781 gaaacctgtc gtgccagcgg atcgacagta ctaagcttta ctagggacag gattggtgac
     6841 agaaaagccc catccttagg cctcctcctt cctagtctcc tgatattggg tctaaccccc
     6901 acctcctgtt aggcagattc cttatctggt gacacacccc catttcctgg agccatctct
     6961 ctccttgcca gaacctctaa ggtttgctta cgatggagcc agagaggatc ctgggaggga
     7021 gagcttggca gggggtggga gggaaggggg ggatgcgtga cctgcccggt tctcagtggc
     7081 caccctgcgc taccctctcc cagaacctga gctgctctga cgcggctgtc tggtgcgttt
     7141 cactgatcct ggtgctgcag cttccttaca cttcccaaga ggagaagcag tttggaaaaa
     7201 caaaatcaga ataagttggt cctgagttct aactttggct cttcaccttt ctagtcccca
     7261 atttatattg ttcctccgtg cgtcagtttt acctgtgaga taaggccagt agccagcccc
     7321 gtcctggcag ggctgtggtg aggagggggg tgtccgtgtg gaaaactccc tttgtgagaa
     7381 tggtgcgtcc taggtgttca ccaggtcgtg gccgcctcta ctccctttct ctttctccat
     7441 ccttctttcc ttaaagagtc cccagtgcta tctgggacat attcctccgc ccagagcagg
     7501 gtcccgcttc cctaaggccc tgctctgggc ttctgggttt gagtccttgg caagcccagg
     7561 agaggcgctc aggcttccct gtcccccttc ctcgtccacc atctcatgcc cctggctctc
     7621 ctgccccttc cctacagggg ttcctggctc tgctctaagg gcgaattcgc ggccgctaaa
     7681 ttcaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt
     7741 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     7801 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagccta
     7861 tacgtacggc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg
     7921 gatgtacaga gtgatattat tgacacgccg gggcgacgga tggtgatccc cctggccagt
     7981 gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat
     8041 gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa
     8101 gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc
     8161 tggggaatat aaatgtcagg catgagatta tcaaaaagga tcttcaccta gatccttttc
     8221 acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct
     8281 atctggacaa gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca
     8341 tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg
     8401 gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca
     8461 aggatctgat ggcgcagggg atcaagctct gatcaagaga caggatgagg atcgtttcgc
     8521 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     8581 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     8641 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     8701 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     8761 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     8821 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     8881 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     8941 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     9001 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     9061 ggcgaggatc tcgtcgtgac ccatgcatgg cgatgcctgc ttgccgaata tcatggtgga
     9121 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     9181 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     9241 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     9301 tcttgacgag ttcttctgaa ttattaacgc ttacaatttc ctgatgcggt attttctcct
     9361 tacgcatctg tgcggtattt cacaccgcat caggtggcac ttttcgggga aatgtgcgcg
     9421 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagattat
     9481 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     9541 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     9601 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     9661 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     9721 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     9781 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     9841 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     9901 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     9961 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
    10021 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
    10081 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga atactcaacc aagtcattct
    10141 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
    10201 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
    10261 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
    10321 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
    10381 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
    10441 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gaccaaaatc ccttaacgtg
    10501 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
    10561 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
    10621 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
    10681 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
    10741 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
    10801 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
    10861 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
    10921 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
    10981 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
    11041 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
    11101 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
    11161 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
    11221 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
    11281 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
    11341 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
    11401 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
    11461 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
    11521 ttcacacagg aaacagctat gaccatgatt acgccaagct cagaattaac cctcactaaa
    11581 ggga
//
