LOCUS       sequence-413999-       14267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains PE2 prime editing gRNA cloning site, inducible prime editor
            2, rtTA-T2A-puro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14267)
  AUTHORS   Nuttle X, Burt ND, Currall B, Moyses-Oliveira M, Mohajeri K, Bhavsar
            R, Lucente D, Yadav R, Tai DJC, Gusella JF, Talkowski ME
  TITLE     Parallelized engineering of mutational models using piggyBac 
            transposon delivery of CRISPR libraries.
  JOURNAL   Cell Rep Methods. 2023 Dec 5:100672. doi: 
            10.1016/j.crmeth.2023.100672.
  PUBMED    38091988
REFERENCE   2  (bases 1 to 14267)
  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: "Cell Rep 
            Methods. 2023 Dec 5:100672. doi: 10.1016/j.crmeth.2023.100672."
COMMENT     Sequence Label: pPB9P2
FEATURES             Location/Qualifiers
     source          1..14267
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..67
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        554..794
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     primer_bind     554..574
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     725..744
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        897..1275
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    905..923
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    941..959
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    977..995
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1013..1031
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1049..1067
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1085..1103
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1121..1139
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1184..1208
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1302..1311
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1314..1334
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1335..3848,3849..3851,3852..5435)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1335..5435
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7608..7628
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(7683..7702)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7727..7775
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        8006..8217
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             8230..8498
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             8532..9275
                     /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             9276..9329
                     /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             9330..9929
                     /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(9330..9349)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     9826..9846
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    9962..10083
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9999..10018)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10053..10072
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   10852..10886
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11377..11393)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11377..11393)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11390..11412)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11401..11417
                     /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(11425..11431,11432..11449,11450..11455))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11470..11491
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11608..11625)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11779..12367)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11859..11878)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12538..13329,13330..13398))
                     /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     13161..13180
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13399..13503)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(13529..13984)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(13666..13687)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     13878..13897
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     14111..14133
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     14125..14142
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     14126..14142
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg tgtaaaattg
       61 acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa
      121 gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt
      181 tatttattta ttaaaaaaaa caaaaactca aaatttcttc tataaagtaa caaaactttt
      241 atgagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg
      301 gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag
      361 ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg
      421 aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaggcctc
      481 cacggccaga ctagaggtac caacgcggcc tttttacggt tcctggcctt ttgctggcct
      541 tttgctcaca tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      601 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
      661 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      721 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      781 cttgtggaaa ggacgaaacg cgtgagacgt tctctgtatg agaccactgt ccacgtctcg
      841 aattcgctag ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagctcgagt
      901 ttactcccta tcagtgatag agaacgtatg aagagtttac tccctatcag tgatagagaa
      961 cgtatgcaga ctttactccc tatcagtgat agagaacgta taaggagttt actccctatc
     1021 agtgatagag aacgtatgac cagtttactc cctatcagtg atagagaacg tatctacagt
     1081 ttactcccta tcagtgatag agaacgtata tccagtttac tccctatcag tgatagagaa
     1141 cgtataagct ttaggcgtgt acggtgggcg cctataaaag cagagctcgt ttagtgaacc
     1201 gtcagatcgc ctggagcaat tccacaacac ttttgtctta taccaacttt ccgtaccact
     1261 tcctaccctc gtaaaggtct agagctagcg agttcgaatt tgccaccatg gccccaaaga
     1321 agaagcggaa agtcgacaag aagtacagca tcggcctgga catcggcacc aactctgtgg
     1381 gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca
     1441 acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg
     1501 aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga
     1561 accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct
     1621 tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc
     1681 ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc
     1741 acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg
     1801 ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg
     1861 acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg
     1921 aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga
     1981 gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc
     2041 tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg
     2101 acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca
     2161 acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt
     2221 ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc
     2281 tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag
     2341 ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga
     2401 acggctacgc cggctacatt gacggcggag ccagccagga agagttctac aagttcatca
     2461 agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg
     2521 acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg
     2581 gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc
     2641 gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca
     2701 ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga
     2761 acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca
     2821 acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt
     2881 acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc
     2941 ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc
     3001 ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact
     3061 ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc
     3121 tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg
     3181 aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga
     3241 aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca
     3301 ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca
     3361 agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga
     3421 tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg
     3481 gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca
     3541 tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg
     3601 agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca
     3661 gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga
     3721 aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc
     3781 agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg
     3841 atgtggacgc tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc
     3901 tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga
     3961 agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt
     4021 tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca
     4081 tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact
     4141 cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca
     4201 ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg
     4261 agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc
     4321 tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg
     4381 acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact
     4441 tcttctacag caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga
     4501 tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg
     4561 gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa
     4621 agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg
     4681 ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc
     4741 ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac
     4801 tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga
     4861 atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca
     4921 agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg
     4981 ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt
     5041 acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc
     5101 tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct
     5161 ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc
     5221 accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca
     5281 atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca
     5341 ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg
     5401 agacacggat cgacctgtct cagctgggag gtgactctgg aggatctagc ggaggatcct
     5461 ctggcagcga gacaccagga acaagcgagt cagcaacacc agagagcagt ggcggcagca
     5521 gcggcggcag cagcacccta aatatagaag atgagtatcg gctacatgag acctcaaaag
     5581 agccagatgt ttctctaggg tccacatggc tgtctgattt tcctcaggcc tgggcggaaa
     5641 ccgggggcat gggactggca gttcgccaag ctcctctgat catacctctg aaagcaacct
     5701 ctacccccgt gtccataaaa caatacccca tgtcacaaga agccagactg gggatcaagc
     5761 cccacataca gagactgttg gaccagggaa tactggtacc ctgccagtcc ccctggaaca
     5821 cgcccctgct acccgttaag aaaccaggga ctaatgatta taggcctgtc caggatctga
     5881 gagaagtcaa caagcgggtg gaagacatcc accccaccgt gcccaaccct tacaacctct
     5941 tgagcgggct cccaccgtcc caccagtggt acactgtgct tgatttaaag gatgcctttt
     6001 tctgcctgag actccacccc accagtcagc ctctcttcgc ctttgagtgg agagatccag
     6061 agatgggaat ctcaggacaa ttgacctgga ccagactccc acagggtttc aaaaacagtc
     6121 ccaccctgtt taatgaggca ctgcacagag acctagcaga cttccggatc cagcacccag
     6181 acttgatcct gctacagtac gtggatgact tactgctggc cgccacttct gagctagact
     6241 gccaacaagg tactcgggcc ctgttacaaa ccctagggaa cctcgggtat cgggcctcgg
     6301 ccaagaaagc ccaaatttgc cagaaacagg tcaagtatct ggggtatctt ctaaaagagg
     6361 gtcagagatg gctgactgag gccagaaaag agactgtgat ggggcagcct actccgaaga
     6421 cccctcgaca actaagggag ttcctaggga aggcaggctt ctgtcgcctc ttcatccctg
     6481 ggtttgcaga aatggcagcc cccctgtacc ctctcaccaa accggggact ctgtttaatt
     6541 ggggcccaga ccaacaaaag gcctatcaag aaatcaagca agctcttcta actgccccag
     6601 ccctggggtt gccagatttg actaagccct ttgaactctt tgtcgacgag aagcagggct
     6661 acgccaaagg tgtcctaacg caaaaactgg gaccttggcg tcggccggtg gcctacctgt
     6721 ccaaaaagct agacccagta gcagctgggt ggcccccttg cctacggatg gtagcagcca
     6781 ttgccgtact gacaaaggat gcaggcaagc taaccatggg acagccacta gtcattctgg
     6841 ccccccatgc agtagaggca ctagtcaaac aaccccccga ccgctggctt tccaacgccc
     6901 ggatgactca ctatcaggcc ttgcttttgg acacggaccg ggtccagttc ggaccggtgg
     6961 tagccctgaa cccggctacg ctgctcccac tgcctgagga agggctgcaa cacaactgcc
     7021 ttgatatcct ggccgaagcc cacggaaccc gacccgacct aacggaccag ccgctcccag
     7081 acgccgacca cacctggtac acggatggaa gcagtctctt acaagaggga cagcgtaagg
     7141 cgggagctgc ggtgaccacc gagaccgagg taatctgggc taaagccctg ccagccggga
     7201 catccgctca gcgggctgaa ctgatagcac tcacccaggc cctaaagatg gcagaaggta
     7261 agaagctaaa tgtttatact gatagccgtt atgcttttgc tactgcccat atccatggag
     7321 aaatatacag aaggcgtggg tggctcacat cagaaggcaa agagatcaaa aataaagacg
     7381 agatcttggc cctactaaaa gccctctttc tgcccaaaag acttagcata atccattgtc
     7441 caggacatca aaagggacac agcgccgagg ctagaggcaa ccggatggct gaccaagcgg
     7501 cccgaaaggc agccatcaca gagactccag acacctctac cctcctcata gaaaattcat
     7561 caccctctgg cggctcaaaa agaaccgccg acggcagcga atttgagccc aagaagaaga
     7621 ggaaagtcta aaagggttcg atccctaccg gttagtaatg agtttaaacg ggggaggcta
     7681 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     7741 cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     7801 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     7861 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     7921 ggcggcaggc cctgccatag cagatctgcg aaatcggatc cagctgaatc taagtggatc
     7981 tgcgatcgct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     8041 ttggggggag gggtcggcaa ttgaacgggt gcctagagaa ggtggcgcgg ggtaaactgg
     8101 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     8161 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacagctg
     8221 aagcttcgag gggctcgcat ctctccttca cgcgcccgcc gccctacctg aggccgccat
     8281 ccacgccggt tgagtcgcgt tctgccgcct cccgcctgtg gtgcctcctg aactgcgtcc
     8341 gccgtctagg taagtttaaa gctcaggtcg agaccgggcc tttgtccggc gctcccttgg
     8401 agcctaccta gactcagccg gctctccacg ctttgcctga ccctgcttgc tcaactctac
     8461 gtctttgttt cgttttctgt tctgcgccgt tacagatcta agctgtgacc ggcgcctact
     8521 ctagagccgc catgtctaga ctggacaaga gcaaagtcat aaactctgct ctggaattac
     8581 tcaatggagt cggtatcgaa ggcctgacga caaggaaact cgctcaaaag ctgggagttg
     8641 agcagcctac cctgtactgg cacgtgaaga acaagcgggc cctgctcgat gccctgccaa
     8701 tcgagatgct ggacaggcat catacccact cctgccccct ggaaggcgag tcatggcaag
     8761 actttctgcg gaacaacgcc aagtcatacc gctgtgctct cctctcacat cgcgacgggg
     8821 ctaaagtgca tctcggcacc cgcccaacag agaaacagta cgaaaccctg gaaaatcagc
     8881 tcgcgttcct gtgtcagcaa ggcttctccc tggagaacgc actgtacgct ctgtccgccg
     8941 tgggccactt tacactgggc tgcgtattgg aggaacagga gcatcaagta gcaaaagagg
     9001 aaagagagac acctaccacc gattctatgc ccccacttct gaaacaagca attgagctgt
     9061 tcgaccggca gggagccgaa cctgccttcc ttttcggcct ggaactaatc atatgtggcc
     9121 tggagaaaca gctaaagtgc gaaagcggcg ggccgaccga cgcccttgac gattttgact
     9181 tagacatgct cccagccgat gcccttgacg actttgacct tgatatgctg cctgctgacg
     9241 ctcttgacga ttttgacctt gacatgctcc ccggggaggg cagaggaagt ctactaacat
     9301 gcggtgacgt ggaggagaat cccggcccta tgaccgagta caagcccacg gtgcgcctcg
     9361 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
     9421 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
     9481 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     9541 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     9601 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     9661 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggagtct
     9721 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     9781 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     9841 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     9901 ggtgcatgac ccgcaagccc ggtgcctgaa atcaacctct ggattacaaa atttgtgggt
     9961 aaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
    10021 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
    10081 ttatcatgtc tggaattgac tcaaatgatg tcaattagtc tatcagaagc tcatctggtc
    10141 tcccttccgg gggacaagac atccctgttt aatatttaaa cagcagtgtt cccaaactgg
    10201 gttcttatat cccttgctct ggtcaaccag gttgcagggt ttcctgtcct cacaggaacg
    10261 aagtccctaa agaaacagtg gcagccaggt ttagccccgg aattgactgg attccttttt
    10321 tagggccgcg gcccattggt atggcttttt ccccgtatcc ccccaggtgt ctgcaggctc
    10381 aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg
    10441 ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc
    10501 cgggcggctc gctgctgcgc cctagcgggg gagggacgta attacatccc tgggggcttt
    10561 gggggggggc tgtccctgat atctataaca agaaaatata tatataataa gttatcacgt
    10621 aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa
    10681 aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt
    10741 accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc
    10801 acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca
    10861 attttacgca gactatcttt ctagggttaa tctagctgca tcaggatcat atcgtcgggt
    10921 cttttttccg gctcagtcat cgcccaagct ggcgctatct gggcatcggg gaggaagaag
    10981 cccgtgcctt ttcccgcgag gttgaagcgg catggaaaga gtttgccgag gatgactgct
    11041 gctgcattga cgttgagcga aaacgcacgt ttaccatgat gattcgggaa ggtgtggcca
    11101 tgcacgcctt taacggtgaa ctgttcgttc aggccacctg ggataccagt tcgtcgcggc
    11161 ttttccggac acagttccgg atggtcagcc cgaagcgcat cagcaacccg aacaataccg
    11221 gcgacagccg gaactgccgt gccggtgtgc agattaatga cagcggtgcg gcgctgggat
    11281 attacgtcag cgaggacggg tatcctggct ggatgccgca gaaatggaca tggatacccc
    11341 gtgagttacc cggcgggcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
    11401 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
    11461 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
    11521 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
    11581 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
    11641 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
    11701 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
    11761 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
    11821 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
    11881 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
    11941 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
    12001 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
    12061 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
    12121 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
    12181 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
    12241 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
    12301 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
    12361 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
    12421 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
    12481 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
    12541 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
    12601 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
    12661 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
    12721 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
    12781 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
    12841 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
    12901 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
    12961 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
    13021 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
    13081 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
    13141 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
    13201 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
    13261 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
    13321 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
    13381 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
    13441 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
    13501 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
    13561 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
    13621 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
    13681 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
    13741 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
    13801 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
    13861 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
    13921 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
    13981 gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
    14041 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
    14101 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc ctcgttcatt
    14161 cacgtttttg aacccgtgga ggacgggcag actcgcggtg caaatgtgtt ttacagcgtg
    14221 atggagcaga tgaagatgct cgacacgctg cagaacacgc agctaga
//
