LOCUS       sequence-269429-       11407 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 11407)
  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 11407)
  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-BtW-eSpCas9
FEATURES             Location/Qualifiers
     source          1..11407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     44..66
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   570..604
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(873..889)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(873..889)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        1145..1393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1145..1165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1316..1335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1414..1489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      2203..2212
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2209..2604
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             2623..2676
                     /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             2686..2709
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2728..2748
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2773..5310,5311..5313,5314..5775,5776..5778,5779..5946,
                     5947..5949,5950..6873)
                     /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             6874..6921
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     misc_feature    6948..7536
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7001..7021)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7419..7430)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    7616..7750
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7640..7659)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7694..7713
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   8529..8591
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8721..8738)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8721..8737)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8730..8752)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8879..9334
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8966..8985)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9176..9197
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9360..9464
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9465..9533,9534..10325)
                     /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(9683..9702)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10496..11084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10985..11004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11238..11255
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11372..11393
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgacc atgattacgc caagcgcgcc cgccgggtaa ctcacggggt
      121 atccatgtcc atttctgcgg catccagcca ggatacccgt cctcgctgac gtaatatccc
      181 agcgccgcac cgctgtcatt aatctgcaca ccggcacggc agttccggct gtcgccggta
      241 ttgttcgggt tgctgatgcg cttcgggctg accatccgga actgtgtccg gaaaagccgc
      301 gacgaactgg tatcccaggt ggcctgaacg aacagttcac cgttaaaggc gtgcatggcc
      361 acaccttccc gaatcatcat ggtaaacgtg cgttttcgct caacgtcaat gcagcagcag
      421 tcatcctcgg caaactcttt ccatgccgct tcaacctcgc gggaaaaggc acgggcttct
      481 tcctccccga tgcccagata gcgccagctt gggcgatgac tgagccggaa aaaagacccg
      541 acgatatgat cctgatgcag ctagattaac cctagaaaga tagtctgcgt aaaattgacg
      601 catgcattct tgaaatattg ctctctcttt ctaaatagcg cgaatccgtc gctgtgcatt
      661 taggacatct cagtcgccgc ttggagctcc cgtgaggcgt gcttgtcaat gcggtaagtg
      721 tcactgattt tgaactataa cgaccgcgtg agtcaaaatg acgcatgatt atcttttacg
      781 tgacttttaa gatttaactc atacgataat tatattgtta tttcatgttc tacttacgtg
      841 ataacttatt atatatatat tttcttgtta tagatccact agttctagag ggacagcccc
      901 cccccaaagc ccccagggat gtaattacgt ccctcccccg ctagggggca gcagcgagcc
      961 gcccggggct ccgctccggt ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg
     1021 gacagcccgg gcacggggaa ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc
     1081 tctttgagcc tgcagacacc tggggggata cggggaaaag gcctccaggg ccgccccctt
     1141 caccgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     1201 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     1261 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     1321 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     1381 aggacgaaac accggagacg ctgacccgtc tctgttttag agctagaaat agcaagttaa
     1441 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt
     1501 tgcctccaag gcctgcagga gtcaatggga aaaacccatt ggagccaagt acactgactc
     1561 aatagggact ttccattggg ttttgcccag tacataaggt caataggggg tgagtcaaca
     1621 ggaaagtccc attggagcca agtacattga gtcaataggg actttccaat gggttttgcc
     1681 cagtacataa ggtcaatggg aggtaagcca atgggttttt cccattactg acatgtatac
     1741 tgagtcatta gggactttcc aatgggtttt gcccagtaca taaggtcaat aggggtgaat
     1801 caacaggaaa gtcccattgg agccaagtac actgagtcaa tagggacttt ccattgggtt
     1861 ttgcccagta caaaaggtca atagggggtg agtcaatggg tttttcccat tattggcaca
     1921 tacataaggt caataggggt gactagtgga gaagagcatg cttgagggct gagtgcccct
     1981 cagtgggcag agagcacatg gcccacagtc cctgagaagt tggggggagg ggtgggcaat
     2041 tgaactggtg cctagagaag gtggggcttg ggtaaactgg gaaagtgatg tggtgtactg
     2101 gctccacctt tttccccagg gtgggggaga accatatata agtgcagtag tctctgtgaa
     2161 cattcggttt agtgaacctg caggtcggcc gccacgaccg gtgccaccat ggccaagcct
     2221 ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa cagcatcccc
     2281 atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg catcttcact
     2341 ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt gctgggcact
     2401 gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa tgagaacagg
     2461 ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca tcctgggatc
     2521 aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg
     2581 ccctctggtt atgtgtggga gggcgctagc cttggctccg gcgagggcag gggaagtctt
     2641 ctaacatgcg gggacgtgga ggaaaatccc ggcccactcg gcatggacta caaggacgac
     2701 gatgacaagc tcgatagcgg catggcccca aagaagaagc ggaaggtcgg tatccacgga
     2761 gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc
     2821 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     2881 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
     2941 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
     3001 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     3061 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
     3121 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
     3181 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     3241 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     3301 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     3361 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     3421 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     3481 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     3541 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     3601 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     3661 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     3721 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     3781 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     3841 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     3901 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     3961 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     4021 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     4081 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     4141 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     4201 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     4261 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     4321 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     4381 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     4441 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     4501 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     4561 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     4621 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     4681 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     4741 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     4801 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     4861 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     4921 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     4981 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     5041 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     5101 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     5161 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     5221 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     5281 gtggaccata tcgtgcctca gagctttctg gccgacgact ccatcgacaa caaggtgctg
     5341 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     5401 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     5461 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     5521 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     5581 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     5641 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     5701 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     5761 atcaaaaagt accctgcgct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     5821 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     5881 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     5941 cggaaggcgc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     6001 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     6061 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     6121 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     6181 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     6241 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     6301 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     6361 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     6421 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     6481 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     6541 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     6601 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     6661 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     6721 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     6781 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     6841 acacggatcg acctgtctca gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc
     6901 ggccaggcaa aaaagaaaaa gtaagagcta gagttcgcgt ctggaacaat caacctctgg
     6961 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     7021 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     7081 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     7141 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     7201 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     7261 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     7321 attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca
     7381 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     7441 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     7501 agacgagtcg gatctccctt tgggccgcct ccccgcctgg aattaattct ggctaagcac
     7561 ttcgtggccg tcgatcgttt aaagggaggt agtgagtcga gatatcgata ctagtgatcc
     7621 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     7681 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     7741 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     7801 ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt atgatcctct
     7861 agagggacag ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg
     7921 ggcagcagcg agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc
     7981 cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga
     8041 acgcttctcg ctgctctttg agcctgcaga cacctggggg gatacgggga aaaagccata
     8101 ccaatggagc tcacggggac agcccccccc caaagccccc agggatgtaa ttacgtccct
     8161 cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg cgctcccccc
     8221 gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg gcacgggatc
     8281 gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg gggatacggg
     8341 gaaaaagaat tcgataaaag ttttgttact ttatagaaga aattttgagt ttttgttttt
     8401 tttaataaat aaataaacat aaataaattg tttgttgaat ttattattag tatgtaagtg
     8461 taaatataat aaaacttaat atctattcaa attaataaat aaacctcgat atacagaccg
     8521 ataaaacaca tgcgtcaatt ttacacatga ttatctttaa cgtacgtcac aatatgatta
     8581 tctttctagg gttaatctag ctgcgtgttc tgcagcgtgt cgagcatctt catctgctcc
     8641 atcacgctgt aaaacacatt tgcaccgcga gtctgcccgt cctccacggg ttcaaaaacg
     8701 tgaatgaacg aggcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     8761 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     8821 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     8881 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     8941 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     9001 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     9061 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     9121 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     9181 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     9241 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     9301 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     9361 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     9421 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     9481 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     9541 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     9601 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     9661 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     9721 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     9781 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     9841 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     9901 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     9961 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
    10021 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
    10081 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
    10141 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
    10201 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
    10261 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
    10321 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
    10381 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
    10441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
    10501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
    10561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
    10621 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
    10681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
    10741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
    10801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
    10861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
    10921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
    10981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
    11041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
    11101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
    11161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
    11221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
    11281 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
    11341 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
    11401 cccaggc
//
