LOCUS       sequence-434050-       13294 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral Pulse-Switch vector for sgRNA expression, Cas9 and sgRNA 
            expression are mutual exclusive; blastiR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13294)
  AUTHORS   Chylinski K, Hubmann M, Hanna RE, Yanchus C, Michlits G, Uijttewaal 
            ECH, Doench J, Schramek D, Elling U
  TITLE     CRISPR-Switch regulates sgRNA activity by Cre recombination for 
            sequential editing of two loci.
  JOURNAL   Nat Commun. 2019 Nov 29;10(1):5454. doi: 10.1038/s41467-019-13403-y.
  PUBMED    31784531
REFERENCE   2  (bases 1 to 13294)
  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: "Nat 
            Commun."; date: "2019-11-29"; volume: "10(1):5454. doi"; pages: " 
            10.1038/s41467-019-13403-y"
COMMENT     Sequence Label: p223-Pulse-Switch
FEATURES             Location/Qualifiers
     source          1..13294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(215..248)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    323..911
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(376..396)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(794..805)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(914..930)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(915..931)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             1436..1616
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(1642..1659)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1648..1872
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1918..2346
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2005..2024)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2215..2236
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2355..2375)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2360..2689
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2540..2675
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2602..2621
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2737..2784
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2803..3177
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3307..3428
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3344..3363)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3398..3417
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3477..3493)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3477..3493)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3490..3512)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3501..3517
                     /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(3525..3531,3532..3549,3550..3555))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3570..3591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3708..3725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3879..4467)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3959..3978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4638..5429,5430..5498))
                     /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     5261..5280
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5499..5603)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5678..5697)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5869..6248
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6250..6448
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6403..6423
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6466..6646
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6693..6818
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7311..7544
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7729..7773
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    8071..8188
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8223..8471
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8394..8413
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(8517..8550)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        8609..8820
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      8839..8848
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             8845..12948
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             8845..12948
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             12949..12996
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             12997..13020
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             13030..13086
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(13087..13294,1..191)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
ORIGIN
        1 ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct
       61 gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg
      121 acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt
      181 gggagggcta agaattcgat atcaagctta tcggataact tcgtatagca tacattatac
      241 gaagttatca gcatagcaag tttaaataag gctagtccgt tatcaacttg aaaaagtggc
      301 accgagtcgg tgcttttttg ctaatcaacc tctggattac aaaatttgtg aaagattgac
      361 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
      421 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
      481 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
      541 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
      601 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
      661 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
      721 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
      781 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
      841 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
      901 cgcctccccg catcgatacc gtcgacctcg agacctagaa aaacatggag caatcacaag
      961 tagcaataca gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggagga
     1021 ggtgggtttt ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgt
     1081 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg
     1141 aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattggca
     1201 gaactacaca ccagggccag ggatcagata tccactgacc tttggatggt gctacaagct
     1261 agtaccagtt gagcaagaga aggtagaaga agccaatgaa ggagagaaca cccgcttgtt
     1321 acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtattag agtggaggtt
     1381 tgacagccgc ctagcatttc atcacatggc ccgagagctg catccggact gtactgggtc
     1441 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     1501 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     1561 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagggc
     1621 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     1681 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     1741 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     1801 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     1861 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     1921 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     1981 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2041 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2101 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2161 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2221 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2281 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2341 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     2401 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     2461 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     2521 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     2581 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     2641 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     2701 agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag
     2761 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt
     2821 gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg
     2881 ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg
     2941 accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg
     3001 tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc
     3061 cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc
     3121 gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac
     3181 gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3241 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3301 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3361 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3421 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     3481 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3541 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3601 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3661 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3721 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3781 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3841 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3901 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3961 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4021 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4081 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4141 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4201 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4261 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4321 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4381 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4441 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4501 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4561 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4621 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4681 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4741 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4801 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4861 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4921 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4981 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5041 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5101 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5161 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5221 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5281 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5341 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5401 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5461 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5521 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5581 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     5641 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     5701 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     5761 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     5821 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     5881 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     5941 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     6001 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     6061 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     6121 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     6181 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     6241 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     6301 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     6361 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     6421 gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta
     6481 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     6541 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     6601 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     6661 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     6721 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     6781 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     6841 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     6901 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     6961 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     7021 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     7081 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     7141 ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat
     7201 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     7261 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     7321 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     7381 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     7441 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     7501 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     7561 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     7621 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     7681 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     7741 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     7801 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     7861 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     7921 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     7981 gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg tgcgccaatt
     8041 ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     8101 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     8161 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt
     8221 ttggttaatt atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     8281 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     8341 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     8401 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     8461 gacgaaacac cggagacgat cagtatacgt aacgtctctg tttaagagct atgctgataa
     8521 cttcgtatag catacattat acgaagttat tttttttttt ttagctaggt cttgaaagga
     8581 gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     8641 aagttggggg gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac
     8701 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     8761 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     8821 gaccggttct agagcgctgc caccatggac aagaagtaca gcatcggcct ggacatcggc
     8881 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc
     8941 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg
     9001 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac
     9061 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag
     9121 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag
     9181 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac
     9241 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg
     9301 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc
     9361 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac
     9421 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg
     9481 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag
     9541 aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc
     9601 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac
     9661 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc
     9721 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc
     9781 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg
     9841 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc
     9901 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc
     9961 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag
    10021 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac
    10081 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc
    10141 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg
    10201 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc
    10261 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc
    10321 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
    10381 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag
    10441 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg
    10501 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
    10561 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
    10621 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
    10681 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
    10741 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
    10801 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
    10861 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
    10921 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
    10981 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
    11041 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
    11101 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
    11161 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
    11221 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
    11281 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
    11341 ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc
    11401 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc
    11461 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt
    11521 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
    11581 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
    11641 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa
    11701 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
    11761 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
    11821 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
    11881 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
    11941 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
    12001 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
    12061 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
    12121 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
    12181 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
    12241 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
    12301 aagtccaaga aactgaagag tgtgaaagag ctactgggga tcaccatcat ggaaagaagc
    12361 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
    12421 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
    12481 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
    12541 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
    12601 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
    12661 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
    12721 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
    12781 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
    12841 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
    12901 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc
    12961 gccacaaaga aggctggaca ggctaagaag aagaaagatt acaaagacga tgacgataag
    13021 ggatccggcg caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct
    13081 ggaccgatgg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag agcaacggct
    13141 acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc tctctctagc
    13201 gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa
    13261 ctcgtggtgc tgggcactgc tgctgctgcg gcag
//
