LOCUS       sequence-220977-       12956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6-sgRNA-EFS-Cas9-T2A-mCherry-P2A-Hygro; wtCas9 without NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12956)
  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 12956)
  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: pBC2102-reci
FEATURES             Location/Qualifiers
     source          1..12956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             233..413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /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    1807..1924
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1953..2201
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1953..1973
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2124..2143
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        2376..2587
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2610..2633
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2634..6734
                     /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             2634..6734
                     /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             6756..6809
                     /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             6810..7517
                     /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(6955..6973)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             7533..7589
                     /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             7590..8615
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     misc_feature    8651..9239
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8704..8724)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9122..9133)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9446..9679
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    9751..9872
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9788..9807)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9842..9861
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      9912..10047
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10094..10113)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10095..10113)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10123..10140)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10123..10139)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10132..10154)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10284..10739
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10371..10390)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10581..10602
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10768..10872
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10873..10941,10942..11733)
                     /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(11091..11110)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11904..12492
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12393..12412
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12646..12663
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    12780..12801
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(12816..12821,12822..12839,12840..12846)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12854..12870
                     /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     12859..12881
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     12878..12894
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12878..12894
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     12913..12933
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        12915..12933
                     /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 gatacaataa acgggtctct
      241 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      301 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      361 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      421 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cctcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
     1741 aggtggagag agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg
     1801 gttaactttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     1861 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     1921 ttttatcgat aacgagacta gcctcgatcg ccgagggcct atttcccatg attccttcat
     1981 atttgcatat acgatacaag gctgttagag agataattag aattaatttg actgtaaaca
     2041 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     2101 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     2161 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggagacgga ggacgacgaa
     2221 cgtctctgtt taagagctat gctggaaaca gcatagcaag tttaaataag gctagtccgt
     2281 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaattcgcta gctaggtctt
     2341 gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt
     2401 ccccgagaag ttggggggag gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg
     2461 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
     2521 accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag
     2581 aacacaggac cggtagcgct cccgggatgg attacaaaga cgatgacgat aaggacaaga
     2641 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg
     2701 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     2761 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     2821 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     2881 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     2941 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     3001 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     3061 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     3121 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     3181 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     3241 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     3301 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc
     3361 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     3421 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     3481 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     3541 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     3601 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     3661 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     3721 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     3781 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     3841 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     3901 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     3961 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     4021 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     4081 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     4141 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     4201 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     4261 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     4321 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     4381 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     4441 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     4501 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     4561 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     4621 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     4681 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     4741 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     4801 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     4861 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     4921 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     4981 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     5041 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     5101 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     5161 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     5221 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     5281 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     5341 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     5401 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     5461 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     5521 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     5581 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     5641 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     5701 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     5761 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     5821 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     5881 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     5941 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     6001 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     6061 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     6121 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     6181 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     6241 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     6301 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     6361 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     6421 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     6481 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     6541 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     6601 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     6661 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     6721 agctgggagg cgacggatcc gaatccggca gtggagaggg cagaggaagt ctgctaacat
     6781 gcggtgacgt cgaggagaat cctggcccaa tggtgagcaa gggcgaggag gataacatgg
     6841 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg
     6901 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     6961 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca
     7021 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
     7081 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     7141 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
     7201 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa aaccatgggc tgggaggcct
     7261 cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga
     7321 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
     7381 ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg
     7441 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca
     7501 tggacgagct gtacaagccc gggggaagcg gagctactaa cttcagcctg ctgaagcagg
     7561 ctggagatgt ggaggagaac cctggaccta tgaaaaagcc tgaactcacc gcgacgtctg
     7621 tcgagaagtt tctgatcgaa aagttcgaca gcgtatccga cctgatgcag ctctcggagg
     7681 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     7741 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     7801 cgctcccgat tccggaagtg cttgacattg gggagttcag cgagagcctg acctattgca
     7861 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     7921 ttctgcagcc ggtcgcggag gcaatggatg cgatcgctgc ggccgatctt agccagacga
     7981 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     8041 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     8101 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     8161 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     8221 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     8281 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     8341 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     8401 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     8461 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     8521 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     8581 gacgccccag cactcgtccg agggcaaagg aataggaatt ctaatgtcga cttcgaaacg
     8641 cgtctggaac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     8701 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     8761 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     8821 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     8881 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     8941 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     9001 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc
     9061 atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     9121 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     9181 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc
     9241 tggaattaat tctgcagtcg agacctagaa aaacatggag caatcacaag tagcaataca
     9301 gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt
     9361 ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
     9421 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     9481 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     9541 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     9601 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     9661 gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt
     9721 gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac
     9781 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     9841 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc
     9901 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     9961 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
    10021 agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgcttt tgcgtcgaaa
    10081 cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca
    10141 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
    10201 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
    10261 cagcctgaat ggcgaatggc gcgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
    10321 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
    10381 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
    10441 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
    10501 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
    10561 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
    10621 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
    10681 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc
    10741 ccaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
    10801 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
    10861 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
    10921 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
    10981 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
    11041 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
    11101 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
    11161 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
    11221 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
    11281 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
    11341 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
    11401 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
    11461 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
    11521 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
    11581 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
    11641 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
    11701 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
    11761 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
    11821 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
    11881 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
    11941 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
    12001 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
    12061 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
    12121 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
    12181 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
    12241 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
    12301 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
    12361 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
    12421 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
    12481 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
    12541 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
    12601 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
    12661 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
    12721 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
    12781 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
    12841 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
    12901 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagc
//
