LOCUS       sequence-225603-       13081 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6-sgRNA-EFS-Cas9-T2A-mCherry-P2A-Hygro; wtCas9 with NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13081)
  AUTHORS   Oakes BL, Nadler DC, Flamholz A, Fellmann C, Staahl BT, Doudna JA, 
            Savage DF
  TITLE     Profiling of engineering hotspots identifies an allosteric 
            CRISPR-Cas9 switch.
  JOURNAL   Nat Biotechnol. 2016 May 2. doi: 10.1038/nbt.3528.
  PUBMED    27136077
REFERENCE   2  (bases 1 to 13081)
  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 
            Biotechnol. 2016 May 2. doi: 10.1038/nbt.3528."
COMMENT     Sequence Label: pBC2101-reci
FEATURES             Location/Qualifiers
     source          1..13081
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /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    1806..1923
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1952..2200
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1952..1972
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2123..2142
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        2375..2586
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2609..2674
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2681..2701
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2726..6826
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2726..6826
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6827..6874
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6896..6949
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             6950..7657
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(7095..7113)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             7673..7729
                     /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             7730..8755
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     misc_feature    8791..9379
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8844..8864)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9262..9273)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9586..9819
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    9891..10012
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9928..9947)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9982..10001
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      10052..10187
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10225..10244)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10226..10244)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10254..10271)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10254..10270)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10263..10285)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10412..10867
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10499..10518)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10709..10730
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10893..10997
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10998..11066,11067..11858)
                     /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     complement(11216..11235)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12029..12617
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12518..12537
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12771..12788
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    12905..12926
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(12941..12946,12947..12964,12965..12971)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12979..12995
                     /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)."
     primer_bind     12984..13006
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     13003..13019
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13003..13019
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     13038..13058
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        13040..13058
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa
     1741 ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg acggtatcgg
     1801 ttaactttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     1861 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     1921 tttatcgatc acgagactag cctcgatcgc cgagggccta tttcccatga ttccttcata
     1981 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
     2041 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     2101 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     2161 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggagacggag gacgacgaac
     2221 gtctctgttt aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt
     2281 atcaacttga aaaagtggca ccgagtcggt gctttttttg aattcgctag ctaggtcttg
     2341 aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc
     2401 cccgagaagt tggggggagg ggtcggcaat tgatccggtg cctagagaag gtggcgcggg
     2461 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     2521 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     2581 acacaggacc ggtagcgctc ccgggatgga ctataaggac cacgacggag actacaagga
     2641 tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga agcggaaggt
     2701 cggtatccac ggagtcccag cagccgacaa gaagtacagc atcggcctgg acatcggcac
     2761 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
     2821 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt
     2881 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
     2941 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt
     3001 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
     3061 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
     3121 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
     3181 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
     3241 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
     3301 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
     3361 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
     3421 gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
     3481 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga
     3541 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
     3601 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
     3661 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac
     3721 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
     3781 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta
     3841 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
     3901 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca
     3961 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
     4021 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
     4081 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
     4141 caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga
     4201 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
     4261 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg
     4321 aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt
     4381 caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga
     4441 gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac
     4501 ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga
     4561 ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga
     4621 ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg
     4681 gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa
     4741 gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt
     4801 catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt
     4861 gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat
     4921 taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg
     4981 gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg
     5041 acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag
     5101 ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct
     5161 gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct
     5221 gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga
     5281 caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga
     5341 agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac
     5401 ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa
     5461 ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca
     5521 gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt
     5581 gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta
     5641 caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt
     5701 gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta
     5761 caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac
     5821 cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc
     5881 caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt
     5941 gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa
     6001 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa
     6061 gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg
     6121 cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa
     6181 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag
     6241 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga
     6301 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat
     6361 gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt
     6421 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga
     6481 gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat
     6541 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc
     6601 ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt
     6661 taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg
     6721 gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac
     6781 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaaaa ggccggcggc
     6841 cacgaaaaag gccggccagg caaaaaagaa aaagggatcc gaatccggca gtggagaggg
     6901 cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccaa tggtgagcaa
     6961 gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga
     7021 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
     7081 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga
     7141 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat
     7201 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     7261 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
     7321 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
     7381 aaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
     7441 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac
     7501 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt
     7561 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg
     7621 ccgccactcc accggcggca tggacgagct gtacaagccc gggggaagcg gagctactaa
     7681 cttcagcctg ctgaagcagg ctggagatgt ggaggagaac cctggaccta tgaaaaagcc
     7741 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtatccga
     7801 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     7861 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     7921 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggagttcag
     7981 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     8041 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gcaatggatg cgatcgctgc
     8101 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     8161 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     8221 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     8281 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     8341 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     8401 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     8461 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     8521 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     8581 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     8641 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     8701 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aataggaatt
     8761 ctaatgtcga cttcgaaacg cgtctggaac aatcaacctc tggattacaa aatttgtgaa
     8821 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     8881 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     8941 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     9001 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     9061 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     9121 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     9181 gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg
     9241 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     9301 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     9361 ctttgggccg cctccccgcc tggaattaat tctgcagtcg agacctagaa aaacatggag
     9421 caatcacaag tagcaataca gcagctacca atgctgattg tgcctggcta gaagcacaag
     9481 aggaggagga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca atgacttaca
     9541 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     9601 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
     9661 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     9721 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     9781 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca tgtcatctta
     9841 ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta
     9901 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     9961 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
    10021 ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
    10081 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
    10141 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc
    10201 aattcgcgaa acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
    10261 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
    10321 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
    10381 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
    10441 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
    10501 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
    10561 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
    10621 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
    10681 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
    10741 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
    10801 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
    10861 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
    10921 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
    10981 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
    11041 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
    11101 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
    11161 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
    11221 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
    11281 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
    11341 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
    11401 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
    11461 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
    11521 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
    11581 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
    11641 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
    11701 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
    11761 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
    11821 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
    11881 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
    11941 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
    12001 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
    12061 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
    12121 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
    12181 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
    12241 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
    12301 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
    12361 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
    12421 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
    12481 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
    12541 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
    12601 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
    12661 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
    12721 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
    12781 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
    12841 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
    12901 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
    12961 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
    13021 atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag
    13081 c
//
