LOCUS       sequence-361256-       13019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBAC-based doxycycline- and trimethoprim-inducible KRAB-dCas9 
            CRISPR interference plasmid for enhancer and promoter repression in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13019)
  AUTHORS   Tang WWC, Castillo-Venzor A, Gruhn WH, Kobayashi T, Penfold CA, 
            Morgan MD, Sun D, Irie N, Surani MA
  TITLE     Sequential enhancer state remodelling defines human germline 
            competence and specification.
  JOURNAL   Nat Cell Biol. 2022 Apr;24(4):448-460. doi: 
            10.1038/s41556-022-00878-z. Epub 2022 Apr 11.
  PUBMED    35411086
REFERENCE   2  (bases 1 to 13019)
  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"; doi: 
            "10.1038/s41556-022-00878-z"; journalName: "Nat Cell Biol"; date: 
            "2022-04"; volume: "24"; issue: "4"; pages: "448-460"
COMMENT     Sequence Label: pPB-Ins-TRE3Gp-KRAB-dCas9-ecDHFR-IRES-GFP-EF1A...
FEATURES             Location/Qualifiers
     source          1..13019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1388..1766
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    1396..1414
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1432..1450
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1468..1486
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1504..1522
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1540..1558
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1576..1594
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1612..1630
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1675..1699
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1774..1783
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1810..1995
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             join(2017..2043,2044..2046,2047..4533,4534..4536,
                     4537..6120)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2017..6120
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6124..6150
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6130..6156
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             6169..6189
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6196..6216
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6238..6714
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="ISLIAALAVDHVIGMETVMPWNLPADLAWFKRNTLNKPVIMGRHT
                     WESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLP
                     KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     misc_feature    join(6721..7295,7296..7298,7299..7307)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(6888..6905)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     7115..7134
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(7308..7310,7311..7313,7314..8027)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(7308..7310,7311..7313,7314..8024)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7614..7633)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7961..7982
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(8073..8090)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8079..8303
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        8349..8560
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             8573..8841
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             8868..9467
                     /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(8868..8887)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     9364..9384
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(9527..9547)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    9589..9710
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9626..9645)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9680..9699
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   10473..10507
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(10998..11014)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10998..11014)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11011..11033)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11022..11038
                     /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(11046..11052,11053..11070,11071..11076))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11091..11112
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11229..11246)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11400..11988)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11480..11499)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12159..12950,12951..13019))
                     /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     12782..12801
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc acggccacta gtgccctttc gtcttcaaga
     1381 attcctcgag tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca
     1441 gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt
     1501 tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac
     1561 gtatctacag tttactccct atcagtgata gagaacgtat atccagttta ctccctatca
     1621 gtgatagaga acgtataagc tttaggcgtg tacggtgggc gcctataaaa gcagagctcg
     1681 tttagtgaac cgtcagatcg cctggagcaa ttccacaaca cttttgtctt ataccaactt
     1741 tccgtaccac ttcctaccct cgtaaaggcg gccgccacca tggatgctaa gtcactaact
     1801 gcctggtccc ggacactggt gaccttcaag gatgtatttg tggacttcac cagggaggag
     1861 tggaagctgc tggacactgc tcagcagatc gtgtacagaa atgtgatgct ggagaactat
     1921 aagaacctgg tttccttggg ttatcagctt actaagccag atgtgatcct ccggttggag
     1981 aagggagaag agcccggcgg ttccggcgga gggtcgatgg acaagaagta cagcatcggc
     2041 ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc
     2101 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
     2161 ggcgccctgc tgttcgacag cggagaaaca gccgaggcca cccggctgaa gagaaccgcc
     2221 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
     2281 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
     2341 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
     2401 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
     2461 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
     2521 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
     2581 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc
     2641 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
     2701 ctgcccggcg agaagaagaa tggcctgttc ggcaacctga ttgccctgag cctgggcctg
     2761 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
     2821 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
     2881 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
     2941 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac
     3001 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
     3061 gagattttct tcgaccagag caagaacggc tacgccggct acatcgatgg cggagccagc
     3121 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
     3181 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
     3241 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat
     3301 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
     3361 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
     3421 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgccagcgcc
     3481 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
     3541 cccaagcaca gcctgctgta cgagtacttc accgtgtaca acgagctgac caaagtgaaa
     3601 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaagccatc
     3661 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac
     3721 ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac
     3781 gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac
     3841 aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac
     3901 agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg
     3961 aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac
     4021 ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc
     4081 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc
     4141 cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc
     4201 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg
     4261 aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag
     4321 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc
     4381 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac
     4441 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg
     4501 gacatcaacc ggctgtccga ctacgatgtg gacgctatcg tgcctcagag ctttctgaag
     4561 gacgactcca tcgataacaa agtgctgact cggagcgaca agaaccgggg caagagcgac
     4621 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcgcca gctgctgaat
     4681 gccaagctga ttacccagag gaagttcgac aatctgacca aggccgagag aggcggcctg
     4741 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca
     4801 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaacgacaaa
     4861 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag
     4921 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac
     4981 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc
     5041 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa
     5101 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc
     5161 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa
     5221 acaggcgaga tcgtgtggga taagggccgg gactttgcca ccgtgcggaa agtgctgtct
     5281 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag
     5341 tctatcctgc ccaagaggaa cagcgacaag ctgatcgcca gaaagaagga ctgggaccct
     5401 aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa
     5461 gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc
     5521 atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa
     5581 gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac
     5641 ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg
     5701 ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc
     5761 cccgaggata atgagcagaa acagctgttt gtggaacagc acaaacacta cctggacgag
     5821 atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac
     5881 aaggtgctga gcgcctacaa caagcacaga gacaagccta tcagagagca ggccgagaat
     5941 atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac
     6001 accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc
     6061 caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac
     6121 gcctatccct atgacgtgcc cgattatgcc agcctgggca gcggctcccc caagaaaaaa
     6181 cgcaaggtgg aagatcctaa gaaaaagcgg aaagtgggat ccgggggagc aggaggcatc
     6241 agtctgattg cggcgttagc ggtagatcac gttatcggca tggaaaccgt catgccgtgg
     6301 aacctgcctg ccgatctcgc ctggtttaaa cgcaacacct taaataaacc cgtgattatg
     6361 ggccgccata cctgggaatc aatcggtcgt ccgttgccag gacgcaaaaa tattatcctc
     6421 agcagtcaac cgagtacgga cgatcgcgta acgtgggtga agtcggtgga tgaagccatc
     6481 gcggcgtgtg gtgacgtacc agaaatcatg gttattggcg gcggtcgcgt ttatgaacag
     6541 ttcttgccaa aagcgcaaaa actgtatctg acgcatatcg acgcagaagt ggaaggcgac
     6601 acccatttcc cggattacga gccggatgac tgggaatcgg tattcagcga attccacgat
     6661 gctgatgcgc agaactctca cagctattgc tttgagattc tggagcggcg atgaaccggt
     6721 cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg
     6781 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg
     6841 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg
     6901 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca
     6961 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct
     7021 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca
     7081 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa
     7141 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggta
     7201 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg
     7261 ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg gtgagcaagg
     7321 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     7381 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     7441 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     7501 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     7561 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     7621 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     7681 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     7741 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     7801 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     7861 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     7921 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     7981 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaggc cgctcgataa
     8041 gcttgatcga tgatctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     8101 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     8161 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     8221 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     8281 tggggatgcg gtgggctcta tggacgcgtg gccgcgaagg atctgcgatc gctccggtgc
     8341 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     8401 caattgaacg ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     8461 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     8521 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc gaggggctcg
     8581 catctctcct tcacgcgccc gccgccctac ctgaggccgc catccacgcc ggttgagtcg
     8641 cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct aggtaagttt
     8701 aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct tggagcctac ctagactcag
     8761 ccggctctcc acgctttgcc tgaccctgct tgctcaactc tacgtctttg tttcgttttc
     8821 tgttctgcgc cgttacagat ccaagctgtg accggcgcct acgctagatg accgagtaca
     8881 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg
     8941 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc
     9001 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     9061 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     9121 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     9181 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     9241 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     9301 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     9361 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     9421 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgagtc gacaatcaac
     9481 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     9541 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc gttaactaaa cttgtttatt
     9601 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     9661 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     9721 aattgactca aatgatgtca attagtctat cagaagctca tctggtctcc cttccggggg
     9781 acaagacatc cctgtttaat atttaaacag cagtgttccc aaactgggtt cttatatccc
     9841 ttgctctggt caaccaggtt gcagggtttc ctgtcctcac aggaacgaag tccctaaaga
     9901 aacagtggca gccaggttta gccccggaat tgactggatt ccttttttag ggcccattgg
     9961 tatggctttt tccccgtatc cccccaggtg tctgcaggct caaagagcag cgagaagcgt
    10021 tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg cacgctgccg
    10081 gctcggggat gcggggggag cgccggaccg gagcggagcc ccgggcggct cgctgctgcc
    10141 ccctagcggg ggagggacgt aattacatcc ctgggggctt tggggggggg ctgtccctga
    10201 tatctataac aagaaaatat atatataata agttatcacg taagtagaac atgaaataac
    10261 aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt
    10321 ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc
    10381 ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct
    10441 atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt
    10501 tctagggtta atctagctgc atcaggatca tatcgtcggg tcttttttcc ggctcagtca
    10561 tcgcccaagc tggcgctatc tgggcatcgg ggaggaagaa gcccgtgcct tttcccgcga
    10621 ggttgaagcg gcatggaaag agtttgccga ggatgactgc tgctgcattg acgttgagcg
    10681 aaaacgcacg tttaccatga tgattcggga aggtgtggcc atgcacgcct ttaacggtga
    10741 actgttcgtt caggccacct gggataccag ttcgtcgcgg cttttccgga cacagttccg
    10801 gatggtcagc ccgaagcgca tcagcaaccc gaacaatacc ggcgacagcc ggaactgccg
    10861 tgccggtgtg cagattaatg acagcggtgc ggcgctggga tattacgtca gcgaggacgg
    10921 gtatcctggc tggatgccgc agaaatggac atggataccc cgtgagttac ccggcgggcg
    10981 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
    11041 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
    11101 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
    11161 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
    11221 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
    11281 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
    11341 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
    11401 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
    11461 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
    11521 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
    11581 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
    11641 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
    11701 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
    11761 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
    11821 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
    11881 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
    11941 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
    12001 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
    12061 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
    12121 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
    12181 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
    12241 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
    12301 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
    12361 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
    12421 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
    12481 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
    12541 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
    12601 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
    12661 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
    12721 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
    12781 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
    12841 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
    12901 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
    12961 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcat
//
