LOCUS       sequence-267549-       11599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac vector for CRISPR/SpCas9 applications (nuclease, base 
            editing, or epigenetic modifications). Provides gRNA and EspCas9 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11599)
  AUTHORS   Fischer AD, Veronese-Paniagua DA, Swaminathan S, Kashima H, Rubin 
            DC, Madison BB
  TITLE     The oncogenic function of PLAGL2 is mediated via ASCL2 and IGF2 and 
            a Wnt-independent mechanism in colorectal cancer.
  JOURNAL   Am J Physiol Gastrointest Liver Physiol. 2023 Jun 13. doi: 
            10.1152/ajpgi.00058.2022.
  PUBMED    37310750
REFERENCE   2  (bases 1 to 11599)
  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: "Am J 
            Physiol Gastrointest Liver Physiol. 2023 Jun 13. doi: 
            10.1152/ajpgi.00058.2022."
COMMENT     Sequence Label: BII-gR-PtW-eSpCas9
FEATURES             Location/Qualifiers
     source          1..11599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..24
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8..24
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   515..549
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(818..834)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(818..834)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        1090..1338
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1090..1110
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1261..1280
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1359..1434
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             2766..2819
                     /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             2826..2852
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             2826..2852
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2865..2885
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2910..5447,5448..5450,5451..5912,5913..5915,5916..6083,
                     6084..6086,6087..7010)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7011..7058
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     misc_feature    7085..7673
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7138..7158)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7556..7567)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    7753..7887
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7777..7796)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7831..7850
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   8666..8728
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8858..8875)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8858..8874)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8867..8889)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9016..9471
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9103..9122)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9313..9334
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9497..9601
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9602..9670,9671..10462)
                     /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(9820..9839)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10633..11221
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11122..11141
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11375..11392
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11509..11530
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11545..11550,11551..11568,11569..11575)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11583..11599
                     /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     join(11588..11599,1..11)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcccgccg ggtaactcac
       61 ggggtatcca tgtccatttc tgcggcatcc agccaggata cccgtcctcg ctgacgtaat
      121 atcccagcgc cgcaccgctg tcattaatct gcacaccggc acggcagttc cggctgtcgc
      181 cggtattgtt cgggttgctg atgcgcttcg ggctgaccat ccggaactgt gtccggaaaa
      241 gccgcgacga actggtatcc caggtggcct gaacgaacag ttcaccgtta aaggcgtgca
      301 tggccacacc ttcccgaatc atcatggtaa acgtgcgttt tcgctcaacg tcaatgcagc
      361 agcagtcatc ctcggcaaac tctttccatg ccgcttcaac ctcgcgggaa aaggcacggg
      421 cttcttcctc cccgatgccc agatagcgcc agcttgggcg atgactgagc cggaaaaaag
      481 acccgacgat atgatcctga tgcagctaga ttaaccctag aaagatagtc tgcgtaaaat
      541 tgacgcatgc attcttgaaa tattgctctc tctttctaaa tagcgcgaat ccgtcgctgt
      601 gcatttagga catctcagtc gccgcttgga gctcccgtga ggcgtgcttg tcaatgcggt
      661 aagtgtcact gattttgaac tataacgacc gcgtgagtca aaatgacgca tgattatctt
      721 ttacgtgact tttaagattt aactcatacg ataattatat tgttatttca tgttctactt
      781 acgtgataac ttattatata tatattttct tgttatagat ccactagttc tagagggaca
      841 gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc
      901 gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt
      961 gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc
     1021 gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaggcctc cagggccgcc
     1081 cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     1141 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     1201 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     1261 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     1321 tggaaaggac gaaacaccgg agacgctgac ccgtctctgt tttagagcta gaaatagcaa
     1381 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     1441 gtttttgcct ccaaggcctg caggagtcaa tgggaaaaac ccattggagc caagtacact
     1501 gactcaatag ggactttcca ttgggttttg cccagtacat aaggtcaata gggggtgagt
     1561 caacaggaaa gtcccattgg agccaagtac attgagtcaa tagggacttt ccaatgggtt
     1621 ttgcccagta cataaggtca atgggaggta agccaatggg tttttcccat tactgacatg
     1681 tatactgagt cattagggac tttccaatgg gttttgccca gtacataagg tcaatagggg
     1741 tgaatcaaca ggaaagtccc attggagcca agtacactga gtcaataggg actttccatt
     1801 gggttttgcc cagtacaaaa ggtcaatagg gggtgagtca atgggttttt cccattattg
     1861 gcacatacat aaggtcaata ggggtgacta gtggagaaga gcatgcttga gggctgagtg
     1921 cccctcagtg ggcagagagc acatggccca cagtccctga gaagttgggg ggaggggtgg
     1981 gcaattgaac tggtgcctag agaaggtggg gcttgggtaa actgggaaag tgatgtggtg
     2041 tactggctcc acctttttcc ccagggtggg ggagaaccat atataagtgc agtagtctct
     2101 gtgaacattc ggtttagtga acctgcaggt cggccgccac gaccggtgcc accatgactg
     2161 aatacaaacc aactgttcgc ctggcaactc gtgatgatgt tccacgtgca gttcgcaccc
     2221 tggctgctgc atttgctgac taccctgcaa cccgtcacac tgtggaccca gaccgccaca
     2281 ttgaacgtgt gactgaactg caggagctgt tcctgacccg tgtgggcctg gacattggca
     2341 aagtgtgggt ggcagatgat ggtgctgctg tggcagtgtg gaccacccct gaatctgttg
     2401 aagctggtgc agtgtttgct gagattggcc cacgcatggc agaactgtct ggcagccgcc
     2461 tggcagcaca acagcagatg gaaggtctgc tggcaccaca ccgcccaaaa gaacctgctt
     2521 ggttcctggc aactgtgggt gtgagccctg accaccaggg taagggcctg ggctctgcag
     2581 tggtgctgcc tggtgtggaa gcagctgaac gtgcaggtgt gcctgctttc ctggagacct
     2641 cagctccacg caacctgcct ttctatgaac gcctgggctt cactgtgact gctgatgtgg
     2701 aagtgccaga aggcccacgc acttggtgca tgactcgcaa accaggtgct agccttggct
     2761 ccggcgaggg caggggaagt cttctaacat gcggggacgt ggaggaaaat cccggcccac
     2821 tcggctaccc ctacgacgtg cccgactacg ccagcggcat ggccccaaag aagaagcgga
     2881 aggtcggtat ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg
     2941 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     3001 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     3061 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     3121 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     3181 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     3241 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     3301 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     3361 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     3421 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     3481 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     3541 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     3601 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
     3661 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     3721 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     3781 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     3841 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     3901 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     3961 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     4021 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     4081 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     4141 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     4201 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     4261 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     4321 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     4381 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     4441 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     4501 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     4561 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     4621 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     4681 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     4741 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     4801 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     4861 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     4921 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     4981 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     5041 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     5101 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     5161 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     5221 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     5281 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     5341 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     5401 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctggcc gacgactcca
     5461 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     5521 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     5581 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     5641 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     5701 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     5761 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     5821 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     5881 tcgtgggaac cgccctgatc aaaaagtacc ctgcgctgga aagcgagttc gtgtacggcg
     5941 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     6001 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     6061 tggccaacgg cgagatccgg aaggcgcctc tgatcgagac aaacggcgaa accggggaga
     6121 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     6181 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     6241 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     6301 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     6361 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     6421 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     6481 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     6541 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     6601 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     6661 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     6721 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     6781 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     6841 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     6901 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     6961 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg
     7021 cggccacgaa aaaggccggc caggcaaaaa agaaaaagta agagctagag ttcgcgtctg
     7081 gaacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     7141 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     7201 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     7261 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     7321 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     7381 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     7441 gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct
     7501 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     7561 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     7621 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcctggaat
     7681 taattctggc taagcacttc gtggccgtcg atcgtttaaa gggaggtagt gagtcgagat
     7741 atcgatacta gtgatccaga catgataaga tacattgatg agtttggaca aaccacaact
     7801 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     7861 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     7921 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg
     7981 gctgattatg atcctctaga gggacagccc ccccccaaag cccccaggga tgtaattacg
     8041 tccctccccc gctagggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc
     8101 cccccgcatc cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg
     8161 ggatcgcttt cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat
     8221 acggggaaaa agccatacca atggagctca cggggacagc ccccccccaa agcccccagg
     8281 gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg gctccgctcc
     8341 ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg
     8401 gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga gcctgcagac
     8461 acctgggggg atacggggaa aaagaattcg ataaaagttt tgttacttta tagaagaaat
     8521 tttgagtttt tgtttttttt aataaataaa taaacataaa taaattgttt gttgaattta
     8581 ttattagtat gtaagtgtaa atataataaa acttaatatc tattcaaatt aataaataaa
     8641 cctcgatata cagaccgata aaacacatgc gtcaatttta cacatgatta tctttaacgt
     8701 acgtcacaat atgattatct ttctagggtt aatctagctg cgtgttctgc agcgtgtcga
     8761 gcatcttcat ctgctccatc acgctgtaaa acacatttgc accgcgagtc tgcccgtcct
     8821 ccacgggttc aaaaacgtga atgaacgagg cgcgctcact ggccgtcgtt ttacaacgtc
     8881 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     8941 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     9001 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     9061 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     9121 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     9181 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     9241 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     9301 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     9361 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     9421 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     9481 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     9541 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     9601 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     9661 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     9721 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     9781 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     9841 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     9901 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     9961 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
    10021 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
    10081 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
    10141 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
    10201 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
    10261 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
    10321 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
    10381 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
    10441 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
    10501 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
    10561 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
    10621 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
    10681 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
    10741 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
    10801 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
    10861 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
    10921 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
    10981 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
    11041 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
    11101 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
    11161 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
    11221 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
    11281 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
    11341 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
    11401 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
    11461 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
    11521 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
    11581 aattgtgagc ggataacaa
//
