LOCUS       sequence-237481-       13029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6-sgRNA EFS-Cas9-wt-P2A-Puro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13029)
  AUTHORS   Oakes BL, Fellmann C, Rishi H, Taylor KL, Ren SM, Nadler DC, Yokoo 
            R, Arkin AP, Doudna JA, Savage DF
  TITLE     CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome
            Modification.
  JOURNAL   Cell. 2019 Jan 10;176(1-2):254-267.e16. doi: 
            10.1016/j.cell.2018.11.052.
  PUBMED    30633905
REFERENCE   2  (bases 1 to 13029)
  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."; 
            date: "2019-01-10"; volume: "176(1-2):254-267.e16. doi"; pages: " 
            10.1016/j.cell.2018.11.052"
COMMENT     Sequence Label: pCF204
FEATURES             Location/Qualifiers
     source          1..13029
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2608..2856
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2608..2628
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2779..2798
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3031..3242
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3261..3270
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3267..7370
                     /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             3267..7370
                     /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             7371..7418
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7419..7442
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7452..7508
                     /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             7509..8105
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     8002..8022
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8121..8709
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8174..8194)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8592..8603)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8781..9014
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9040..9057)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9046..9270
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9316..9744
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9403..9422)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9613..9634
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9753..9773)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9758..10087
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9938..10073
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10000..10019
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10135..10182
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10201..10575
                     /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    10705..10826
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10742..10761)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10796..10815
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10875..10891)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10875..10891)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10888..10910)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10899..10915
                     /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(10923..10929,10930..10947,10948..10953))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10968..10989
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11106..11123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11277..11865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11357..11376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12036..12827,12828..12896))
                     /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     12659..12678
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12897..13001)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg
     2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2821 ttggctttat atatcttgtg gaaaggacga aacaccggag acggaggacg acgaacgtct
     2881 ctgtttaaga gctatgctgg aaacagcata gcaagtttaa ataaggctag tccgttatca
     2941 acttgaaaaa gtggcaccga gtcggtgctt tttttgaatt cgctagctag gtcttgaaag
     3001 gagtgggaat tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
     3061 agaagttggg gggaggggtc ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa
     3121 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
     3181 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
     3241 aggaccggtt ctagagcgct gccaccatgg acaagaagta cagcatcggc ctggacatcg
     3301 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     3361 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     3421 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     3481 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     3541 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     3601 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     3661 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     3721 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     3781 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     3841 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     3901 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     3961 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
     4021 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     4081 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     4141 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     4201 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     4261 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     4321 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     4381 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     4441 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     4501 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     4561 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     4621 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     4681 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     4741 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     4801 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     4861 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     4921 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     4981 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     5041 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     5101 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     5161 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     5221 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     5281 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     5341 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     5401 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     5461 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     5521 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     5581 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     5641 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     5701 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     5761 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
     5821 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     5881 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     5941 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     6001 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     6061 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     6121 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     6181 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     6241 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     6301 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     6361 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     6421 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     6481 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     6541 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     6601 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     6661 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     6721 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     6781 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     6841 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     6901 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     6961 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     7021 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     7081 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     7141 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     7201 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     7261 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     7321 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac aagcgacctg
     7381 ccgccacaaa gaaggctgga caggctaaga agaagaaaga ttacaaagac gatgacgata
     7441 agggatccgg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc
     7501 ctggaccgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
     7561 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc
     7621 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     7681 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     7741 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     7801 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     7861 aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc
     7921 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     7981 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     8041 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     8101 cctgaacgcg ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg
     8161 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     8221 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     8281 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     8341 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     8401 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     8461 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     8521 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     8581 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     8641 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     8701 cctccccgcg tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     8761 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct
     8821 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     8881 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     8941 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     9001 gaaaatctct agcagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     9061 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     9121 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     9181 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     9241 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     9301 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     9361 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     9421 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     9481 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     9541 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     9601 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     9661 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     9721 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     9781 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     9841 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     9901 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     9961 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
    10021 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
    10081 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac
    10141 aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc
    10201 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc
    10261 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt
    10321 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac
    10381 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag
    10441 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag
    10501 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc
    10561 gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg
    10621 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
    10681 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
    10741 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
    10801 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
    10861 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
    10921 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
    10981 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
    11041 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
    11101 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
    11161 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
    11221 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
    11281 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
    11341 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
    11401 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
    11461 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
    11521 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
    11581 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
    11641 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
    11701 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
    11761 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
    11821 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
    11881 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
    11941 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
    12001 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
    12061 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
    12121 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
    12181 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
    12241 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
    12301 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
    12361 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
    12421 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
    12481 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
    12541 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
    12601 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
    12661 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
    12721 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
    12781 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
    12841 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
    12901 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
    12961 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
    13021 ccacctgac
//
