LOCUS       sequence-414005-       12842 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains Cas9 gRNA cloning site, inducible Cas9, UCOE, 
            puro-T2A-eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12842)
  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 12842)
  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: pPB9
FEATURES             Location/Qualifiers
     source          1..12842
                     /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..802
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     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"
     misc_RNA        821..896
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        960..1338
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    968..986
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1004..1022
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1040..1058
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1076..1094
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1112..1130
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1148..1166
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1184..1202
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1247..1271
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1365..1374
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1377..1397
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1422..5522
                     /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
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1422..5522
                     /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
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5523..5570
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5625..5644)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5669..5717
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        6605..6816
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             6829..7097
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             7131..7727
                     /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(7131..7150)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7627..7647
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             7728..7781
                     /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             join(7782..7784,7785..8498)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(7782..7784,7785..8495)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(7824..7845)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(8085..8104)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8432..8453
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    8537..8658
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8574..8593)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8628..8647
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   9427..9461
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9952..9968)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9952..9968)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9965..9987)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9976..9992
                     /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(10000..10006,10007..10024,10025..10030))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10045..10066
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10183..10200)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10354..10942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10434..10453)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11113..11904,11905..11973))
                     /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     11736..11755
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11974..12078)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(12104..12559)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12241..12262)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12453..12472
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12686..12708
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12700..12717
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12701..12717
                     /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 ggacgaaaca ccgggtcttc gagaagacct gttttagagc tagaaatagc
      841 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
      901 ttgaattcgc tagctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagctcg
      961 agtttactcc ctatcagtga tagagaacgt atgaagagtt tactccctat cagtgataga
     1021 gaacgtatgc agactttact ccctatcagt gatagagaac gtataaggag tttactccct
     1081 atcagtgata gagaacgtat gaccagttta ctccctatca gtgatagaga acgtatctac
     1141 agtttactcc ctatcagtga tagagaacgt atatccagtt tactccctat cagtgataga
     1201 gaacgtataa gctttaggcg tgtacggtgg gcgcctataa aagcagagct cgtttagtga
     1261 accgtcagat cgcctggagc aattccacaa cacttttgtc ttataccaac tttccgtacc
     1321 acttcctacc ctcgtaaagg tctagagcta gcgaattcga atttgccacc atggccccaa
     1381 agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag tactccattg
     1441 ggctcgatat cggcacaaac agcgtcggct gggccgtcat tacggacgag tacaaggtgc
     1501 cgagcaaaaa attcaaagtt ctgggcaata ccgatcgcca cagcataaag aagaacctca
     1561 ttggcgccct cctgttcgac tccggggaga cggccgaagc cacgcggctc aaaagaacag
     1621 cacggcgcag atatacccgc agaaagaatc ggatctgcta cctgcaggag atcttcagca
     1681 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     1741 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     1801 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     1861 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     1921 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     1981 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     2041 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     2101 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     2161 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     2221 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     2281 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     2341 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     2401 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     2461 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     2521 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     2581 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     2641 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     2701 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     2761 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     2821 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     2881 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     2941 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     3001 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     3061 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     3121 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     3181 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     3241 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     3301 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     3361 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     3421 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     3481 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     3541 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     3601 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     3661 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     3721 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     3781 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     3841 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     3901 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga
     3961 aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     4021 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     4081 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     4141 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     4201 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     4261 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     4321 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     4381 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
     4441 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     4501 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     4561 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
     4621 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     4681 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
     4741 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
     4801 ccaagaaata cggcggattc gattctccta cagtcgctta cagtgtactg gttgtggcca
     4861 aagtggagaa agggaagtct aaaaaactca aaagcgtcaa ggaactgctg ggcatcacaa
     4921 tcatggagcg atcaagcttc gaaaaaaacc ccatcgactt tctcgaggcg aaaggatata
     4981 aagaggtcaa aaaagacctc atcattaagc ttcccaagta ctctctcttt gagcttgaaa
     5041 acggccggaa acgaatgctc gctagtgcgg gcgagctgca gaaaggtaac gagctggcac
     5101 tgccctctaa atacgttaat ttcttgtatc tggccagcca ctatgaaaag ctcaaagggt
     5161 ctcccgaaga taatgagcag aagcagctgt tcgtggaaca acacaaacac taccttgatg
     5221 agatcatcga gcaaataagc gaattctcca aaagagtgat cctcgccgac gctaacctcg
     5281 ataaggtgct ttctgcttac aataagcaca gggataagcc catcagggag caggcagaaa
     5341 acattatcca cttgtttact ctgaccaact tgggcgcgcc tgcagccttc aagtacttcg
     5401 acaccaccat agacagaaag cggtacacct ctacaaagga ggtcctggac gccacactga
     5461 ttcatcagtc aattacgggg ctctatgaaa caagaatcga cctctctcag ctcggtggag
     5521 acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag tgaaagggtt
     5581 cgatccctac cggttagtaa tgagtttaaa cgggggaggc taactgaaac acggaaggag
     5641 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggg
     5701 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     5761 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     5821 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     5881 agcagatctg cgaaatcgga tcccgggagg tggtccctgc agttacgcca atgataaccc
     5941 ccgccagaaa aatcttagta gccttccctt tttgttttcc gtgccccaac tcggcggatt
     6001 gactcggccc cttccggaaa cacccgaatc aacttctagt caaattattg ttcacgccgc
     6061 aatgacccac ccctggcccg cgtctgtgga actgacccct ggtgtacagg agagttcgct
     6121 gctgaaagtg gtcccaaagg ggtactagtt tttaagctcc caactccccc tcccccagcg
     6181 tctggaggat tccacaccct cgcaccgcag gggcgaggaa gtgggcggag tccggttttg
     6241 gcgccagccg ctgaggctgc caagcagaaa agccaccgct gaggagactc cggtcactgt
     6301 cctcgccccg cctccccctt ccctcccctt ggggaccacc gggcgccacg ccgcgaacgg
     6361 taagtgccgc ggtcgtcggc gcctccgccc tccccctagg gccccaattc ccagcgggcg
     6421 cggcgcgcgg cccctccccc cgccgggcgc gcgcccgctg ccccgccctt cgtggccgcc
     6481 cggcgtgggc ggtgccaccc ctccccccgg cggccccgcg cgcagctccc ggctccctcc
     6541 cccttcggat gtggcttgag ctgtaggcgc ggagggatct gcgatcgctc cggtgcccgt
     6601 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
     6661 tgaacgggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
     6721 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
     6781 gttctttttc gcaacgggtt tgccgccaga acacagctga agcttcgagg ggctcgcatc
     6841 tctccttcac gcgcccgccg ccctacctga ggccgccatc cacgccggtt gagtcgcgtt
     6901 ctgccgcctc ccgcctgtgg tgcctcctga actgcgtccg ccgtctaggt aagtttaaag
     6961 ctcaggtcga gaccgggcct ttgtccggcg ctcccttgga gcctacctag actcagccgg
     7021 ctctccacgc tttgcctgac cctgcttgct caactctacg tctttgtttc gttttctgtt
     7081 ctgcgccgtt acagatctaa gctgtgaccg gcgcctactc tagagccgcc atgaccgagt
     7141 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     7201 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac cgccacatcg
     7261 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     7321 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     7381 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     7441 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     7501 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     7561 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     7621 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     7681 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgccgag ggcagaggaa
     7741 gtctactaac atgcggtgac gtggaggaga atcccggccc tgtgagcaag ggcgaggagc
     7801 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     7861 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     7921 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     7981 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     8041 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     8101 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     8161 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     8221 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     8281 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     8341 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     8401 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     8461 ccgggatcac tctcggcatg gacgagctgt acaagtgagt cgacaatcaa cctctggatt
     8521 acaaaatttg tgggttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     8581 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     8641 actcatcaat gtatcttatc atgtctggaa ttgactcaaa tgatgtcaat tagtctatca
     8701 gaagctatct ggtctccctt ccgggggaca agacatccct gtttaatatt taaacagcag
     8761 tgttcccaaa ctgggttctt atatcccttg ctctggtcaa ccaggttgca gggtttcctg
     8821 tcctcacagg aacgaagtcc ctaaagaaac agtggcagcc aggtttagcc ccggaattga
     8881 ctggattcct tttttagcgg ccgcggccca ttggtatggc tttttccccg tatcccccca
     8941 ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc
     9001 ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg
     9061 accggagcgg agccccgggc ggctcgctgc tgccccctag cgggggaggg acgtaattac
     9121 atccctgggg gctttggggg ggggctgtcc ctgatatcta taacaagaaa atatatatat
     9181 aataagttat cacgtaagta gaacatgaaa taacaatata attatcgtat gagttaaatc
     9241 ttaaaagtca cgtaaaagat aatcatgcgt cattttgact cacgcggtcg ttatagttca
     9301 aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa gcggcgactg
     9361 agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag caatatttca
     9421 agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaatctag ctgcatcagg
     9481 atcatatcgt cgggtctttt ttccggctca gtcatcgccc aagctggcgc tatctgggca
     9541 tcggggagga agaagcccgt gccttttccc gcgaggttga agcggcatgg aaagagtttg
     9601 ccgaggatga ctgctgctgc attgacgttg agcgaaaacg cacgtttacc atgatgattc
     9661 gggaaggtgt ggccatgcac gcctttaacg gtgaactgtt cgttcaggcc acctgggata
     9721 ccagttcgtc gcggcttttc cggacacagt tccggatggt cagcccgaag cgcatcagca
     9781 acccgaacaa taccggcgac agccggaact gccgtgccgg tgtgcagatt aatgacagcg
     9841 gtgcggcgct gggatattac gtcagcgagg acgggtatcc tggctggatg ccgcagaaat
     9901 ggacatggat accccgtgag ttacccggcg ggcgcgcttg gcgtaatcat ggtcatagct
     9961 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
    10021 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
    10081 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
    10141 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
    10201 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
    10261 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
    10321 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
    10381 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
    10441 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
    10501 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
    10561 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
    10621 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
    10681 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
    10741 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
    10801 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
    10861 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
    10921 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
    10981 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
    11041 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
    11101 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
    11161 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
    11221 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
    11281 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
    11341 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
    11401 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
    11461 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
    11521 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
    11581 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
    11641 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
    11701 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
    11761 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
    11821 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
    11881 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
    11941 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
    12001 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
    12061 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
    12121 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
    12181 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
    12241 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
    12301 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
    12361 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
    12421 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
    12481 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
    12541 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
    12601 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
    12661 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag
    12721 cgcgcctcgt tcattcacgt ttttgaaccc gtggaggacg ggcagactcg cggtgcaaat
    12781 gtgttttaca gcgtgatgga gcagatgaag atgctcgaca cgctgcagaa cacgcagcta
    12841 ga
//
