LOCUS       sequence-414000-       14082 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains cloning site for PE3/3b prime editing gRNA constructs, 
            inducible prime editor 2, rtTA-T2A-puro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14082)
  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 14082)
  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: pPB9P3
FEATURES             Location/Qualifiers
     source          1..14082
                     /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)"
     misc_RNA        573..648
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        712..1090
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    720..738
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    756..774
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    792..810
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    828..846
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    864..882
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    900..918
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    936..954
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     999..1023
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1117..1126
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1129..1149
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1150..3663,3664..3666,3667..5250)
                     /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             1150..5250
                     /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             7423..7443
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(7498..7517)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7542..7590
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        7821..8032
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             8045..8313
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             8347..9090
                     /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             9091..9144
                     /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             9145..9744
                     /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(9145..9164)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     9641..9661
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    9777..9898
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9814..9833)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9868..9887
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   10667..10701
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11192..11208)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11192..11208)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11205..11227)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11216..11232
                     /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(11240..11246,11247..11264,11265..11270))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11285..11306
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11423..11440)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11594..12182)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11674..11693)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12353..13144,13145..13213))
                     /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     12976..12995
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13214..13318)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(13344..13799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(13481..13502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     13693..13712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     13926..13948
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     13940..13957
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     13941..13957
                     /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 tgtgagacgg tcgtctcgaa ttgttttaga gctagaaata gcaagttaaa
      601 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgaattc
      661 gctagctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagct cgagtttact
      721 ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata gagaacgtat
      781 gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc ctatcagtga
      841 tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct acagtttact
      901 ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata gagaacgtat
      961 aagctttagg cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt gaaccgtcag
     1021 atcgcctgga gcaattccac aacacttttg tcttatacca actttccgta ccacttccta
     1081 ccctcgtaaa ggtctagagc tagcgagttc gaatttgcca ccatggcccc aaagaagaag
     1141 cggaaagtcg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
     1201 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     1261 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     1321 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     1381 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     1441 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     1501 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     1561 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     1621 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     1681 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     1741 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     1801 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     1861 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     1921 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     1981 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     2041 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     2101 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     2161 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     2221 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     2281 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     2341 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     2401 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     2461 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     2521 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     2581 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     2641 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     2701 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     2761 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     2821 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     2881 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     2941 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     3001 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     3061 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     3121 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     3181 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     3241 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     3301 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     3361 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     3421 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     3481 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     3541 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     3601 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     3661 gacgctatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
     3721 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     3781 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     3841 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     3901 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     3961 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     4021 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     4081 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     4141 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     4201 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     4261 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     4321 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     4381 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     4441 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     4501 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     4561 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     4621 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     4681 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     4741 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     4801 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     4861 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     4921 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     4981 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     5041 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     5101 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     5161 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     5221 cggatcgacc tgtctcagct gggaggtgac tctggaggat ctagcggagg atcctctggc
     5281 agcgagacac caggaacaag cgagtcagca acaccagaga gcagtggcgg cagcagcggc
     5341 ggcagcagca ccctaaatat agaagatgag tatcggctac atgagacctc aaaagagcca
     5401 gatgtttctc tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg
     5461 ggcatgggac tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc
     5521 cccgtgtcca taaaacaata ccccatgtca caagaagcca gactggggat caagccccac
     5581 atacagagac tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc
     5641 ctgctacccg ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa
     5701 gtcaacaagc gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc
     5761 gggctcccac cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc
     5821 ctgagactcc accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg
     5881 ggaatctcag gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc
     5941 ctgtttaatg aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg
     6001 atcctgctac agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa
     6061 caaggtactc gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag
     6121 aaagcccaaa tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag
     6181 agatggctga ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct
     6241 cgacaactaa gggagttcct agggaaggca ggcttctgtc gcctcttcat ccctgggttt
     6301 gcagaaatgg cagcccccct gtaccctctc accaaaccgg ggactctgtt taattggggc
     6361 ccagaccaac aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg
     6421 gggttgccag atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc
     6481 aaaggtgtcc taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa
     6541 aagctagacc cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc
     6601 gtactgacaa aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc
     6661 catgcagtag aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg
     6721 actcactatc aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc
     6781 ctgaacccgg ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat
     6841 atcctggccg aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc
     6901 gaccacacct ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga
     6961 gctgcggtga ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc
     7021 gctcagcggg ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag
     7081 ctaaatgttt atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata
     7141 tacagaaggc gtgggtggct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc
     7201 ttggccctac taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga
     7261 catcaaaagg gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga
     7321 aaggcagcca tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc
     7381 tctggcggct caaaaagaac cgccgacggc agcgaatttg agcccaagaa gaagaggaaa
     7441 gtctaaaagg gttcgatccc taccggttag taatgagttt aaacggggga ggctaactga
     7501 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
     7561 taaaacgcac gggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     7621 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     7681 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     7741 caggccctgc catagcagat ctgcgaaatc ggatccagct gaatctaagt ggatctgcga
     7801 tcgctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
     7861 gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa actgggaaag
     7921 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
     7981 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac agctgaagct
     8041 tcgaggggct cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg
     8101 ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt
     8161 ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct
     8221 acctagactc agccggctct ccacgctttg cctgaccctg cttgctcaac tctacgtctt
     8281 tgtttcgttt tctgttctgc gccgttacag atctaagctg tgaccggcgc ctactctaga
     8341 gccgccatgt ctagactgga caagagcaaa gtcataaact ctgctctgga attactcaat
     8401 ggagtcggta tcgaaggcct gacgacaagg aaactcgctc aaaagctggg agttgagcag
     8461 cctaccctgt actggcacgt gaagaacaag cgggccctgc tcgatgccct gccaatcgag
     8521 atgctggaca ggcatcatac ccactcctgc cccctggaag gcgagtcatg gcaagacttt
     8581 ctgcggaaca acgccaagtc ataccgctgt gctctcctct cacatcgcga cggggctaaa
     8641 gtgcatctcg gcacccgccc aacagagaaa cagtacgaaa ccctggaaaa tcagctcgcg
     8701 ttcctgtgtc agcaaggctt ctccctggag aacgcactgt acgctctgtc cgccgtgggc
     8761 cactttacac tgggctgcgt attggaggaa caggagcatc aagtagcaaa agaggaaaga
     8821 gagacaccta ccaccgattc tatgccccca cttctgaaac aagcaattga gctgttcgac
     8881 cggcagggag ccgaacctgc cttccttttc ggcctggaac taatcatatg tggcctggag
     8941 aaacagctaa agtgcgaaag cggcgggccg accgacgccc ttgacgattt tgacttagac
     9001 atgctcccag ccgatgccct tgacgacttt gaccttgata tgctgcctgc tgacgctctt
     9061 gacgattttg accttgacat gctccccggg gagggcagag gaagtctact aacatgcggt
     9121 gacgtggagg agaatcccgg ccctatgacc gagtacaagc ccacggtgcg cctcgccacc
     9181 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     9241 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     9301 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     9361 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     9421 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     9481 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc
     9541 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     9601 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     9661 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     9721 atgacccgca agcccggtgc ctgaaatcaa cctctggatt acaaaatttg tgggtaaact
     9781 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     9841 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     9901 atgtctggaa ttgactcaaa tgatgtcaat tagtctatca gaagctcatc tggtctccct
     9961 tccgggggac aagacatccc tgtttaatat ttaaacagca gtgttcccaa actgggttct
    10021 tatatccctt gctctggtca accaggttgc agggtttcct gtcctcacag gaacgaagtc
    10081 cctaaagaaa cagtggcagc caggtttagc cccggaattg actggattcc ttttttaggg
    10141 ccgcggccca ttggtatggc tttttccccg tatcccccca ggtgtctgca ggctcaaaga
    10201 gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc
    10261 cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc
    10321 ggctcgctgc tgcgccctag cgggggaggg acgtaattac atccctgggg gctttggggg
    10381 ggggctgtcc ctgatatcta taacaagaaa atatatatat aataagttat cacgtaagta
    10441 gaacatgaaa taacaatata attatcgtat gagttaaatc ttaaaagtca cgtaaaagat
    10501 aatcatgcgt cattttgact cacgcggtcg ttatagttca aaatcagtga cacttaccgc
    10561 attgacaagc acgcctcacg ggagctccaa gcggcgactg agatgtccta aatgcacagc
    10621 gacggattcg cgctatttag aaagagagag caatatttca agaatgcatg cgtcaatttt
    10681 acgcagacta tctttctagg gttaatctag ctgcatcagg atcatatcgt cgggtctttt
    10741 ttccggctca gtcatcgccc aagctggcgc tatctgggca tcggggagga agaagcccgt
    10801 gccttttccc gcgaggttga agcggcatgg aaagagtttg ccgaggatga ctgctgctgc
    10861 attgacgttg agcgaaaacg cacgtttacc atgatgattc gggaaggtgt ggccatgcac
    10921 gcctttaacg gtgaactgtt cgttcaggcc acctgggata ccagttcgtc gcggcttttc
    10981 cggacacagt tccggatggt cagcccgaag cgcatcagca acccgaacaa taccggcgac
    11041 agccggaact gccgtgccgg tgtgcagatt aatgacagcg gtgcggcgct gggatattac
    11101 gtcagcgagg acgggtatcc tggctggatg ccgcagaaat ggacatggat accccgtgag
    11161 ttacccggcg ggcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
    11221 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
    11281 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
    11341 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
    11401 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
    11461 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
    11521 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
    11581 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
    11641 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
    11701 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
    11761 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
    11821 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
    11881 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
    11941 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
    12001 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
    12061 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
    12121 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
    12181 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
    12241 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
    12301 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
    12361 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
    12421 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
    12481 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
    12541 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
    12601 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
    12661 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
    12721 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
    12781 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
    12841 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
    12901 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
    12961 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
    13021 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
    13081 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
    13141 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
    13201 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
    13261 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
    13321 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
    13381 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
    13441 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
    13501 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
    13561 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
    13621 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
    13681 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
    13741 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
    13801 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
    13861 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
    13921 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcctcgt tcattcacgt
    13981 ttttgaaccc gtggaggacg ggcagactcg cggtgcaaat gtgttttaca gcgtgatgga
    14041 gcagatgaag atgctcgaca cgctgcagaa cacgcagcta ga
//
