LOCUS       sequence-224904-       12940 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 12940)
  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 12940)
  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..12940
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..118
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        147..395
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     147..167
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     318..337
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        570..781
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             804..827
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             828..4928
                     /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             828..4928
                     /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             4950..5003
                     /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             5004..5711
                     /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(5149..5167)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             5727..5783
                     /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             5784..6809
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     misc_feature    6845..7433
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6898..6918)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7316..7327)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7640..7873
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7945..8066
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7982..8001)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8036..8055
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8106..8241
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8279..8298)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8280..8298)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8308..8325)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8308..8324)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8317..8339)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8466..8921
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8553..8572)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8763..8784
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        8947..9051
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9052..9120,9121..9912)
                     /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(9270..9289)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10083..10671
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10572..10591
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10825..10842
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    10959..10980
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(10995..11000,11001..11018,11019..11025)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11033..11049
                     /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     11038..11060
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11057..11073
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11057..11073
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     11092..11112
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        11094..11112
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        11140..11366
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             11367..11547
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11594..11719
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12212..12445
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12630..12674
                     /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"
ORIGIN
        1 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
       61 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttat
      121 cgatcacgag actagcctcg atcgccgagg gcctatttcc catgattcct tcatatttgc
      181 atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga
      241 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
      301 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
      361 tggctttata tatcttgtgg aaaggacgaa acaccggaga cggaggacga cgaacgtctc
      421 tgtttaagag ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa
      481 cttgaaaaag tggcaccgag tcggtgcttt ttttgaattc gctagctagg tcttgaaagg
      541 agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
      601 gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc gcggggtaaa
      661 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
      721 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
      781 ggaccggtag cgctcccggg atggattaca aagacgatga cgataaggac aagaagtaca
      841 gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca
      901 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
      961 acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga
     1021 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct
     1081 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc
     1141 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg
     1201 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca
     1261 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg
     1321 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
     1381 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg
     1441 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga
     1501 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc
     1561 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc
     1621 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
     1681 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc
     1741 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg
     1801 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga
     1861 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg
     1921 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca
     1981 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg
     2041 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc
     2101 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct
     2161 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga
     2221 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg
     2281 cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga
     2341 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca
     2401 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
     2461 aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag
     2521 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc
     2581 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact
     2641 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt
     2701 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca
     2761 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc
     2821 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg
     2881 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag
     2941 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca
     3001 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg
     3061 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag
     3121 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag
     3181 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc
     3241 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc
     3301 aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct
     3361 ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca
     3421 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc
     3481 tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag
     3541 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc
     3601 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga
     3661 atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt
     3721 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg
     3781 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa
     3841 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg
     3901 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt
     3961 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa
     4021 acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag
     4081 tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca
     4141 gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact
     4201 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg
     4261 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga
     4321 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg
     4381 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc
     4441 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac
     4501 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga
     4561 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc
     4621 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta
     4681 atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg
     4741 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt
     4801 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca
     4861 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg
     4921 gaggcgacgg atccgaatcc ggcagtggag agggcagagg aagtctgcta acatgcggtg
     4981 acgtcgagga gaatcctggc ccaatggtga gcaagggcga ggaggataac atggccatca
     5041 tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg
     5101 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
     5161 tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg
     5221 gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc
     5281 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga
     5341 cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca
     5401 acttcccctc cgacggcccc gtaatgcaga agaaaaccat gggctgggag gcctcctccg
     5461 agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga
     5521 aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc
     5581 agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact
     5641 acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg
     5701 agctgtacaa gcctggggga agcggagcta ctaacttcag cctgctgaag caggctggag
     5761 atgtggagga gaaccctgga cctatgaaaa agcctgaact caccgcgacg tctgtcgaga
     5821 agtttctgat cgaaaagttc gacagcgtat ccgacctgat gcagctctcg gagggcgaag
     5881 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
     5941 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
     6001 cgattccgga agtgcttgac attggggagt tcagcgagag cctgacctat tgcatctccc
     6061 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
     6121 agccggtcgc ggaggcaatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
     6181 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
     6241 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
     6301 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
     6361 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
     6421 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
     6481 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
     6541 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
     6601 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
     6661 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
     6721 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
     6781 ccagcactcg tccgagggca aaggaatagg aattctaatg tcgacttcga aacgcgtctg
     6841 gaacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     6901 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     6961 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     7021 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     7081 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     7141 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     7201 gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct
     7261 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     7321 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     7381 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcctggaat
     7441 taattctgca gtcgagacct agaaaaacat ggagcaatca caagtagcaa tacagcagct
     7501 accaatgctg attgtgcctg gctagaagca caagaggagg aggaggtggg ttttccagtc
     7561 acacctcagg tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     7621 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
     7681 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     7741 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     7801 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     7861 aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     7921 aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa
     7981 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     8041 ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc
     8101 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     8161 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     8221 gaggaggctt ttttggaggc ctagggacgt acccaattcg cgaaacgtac ccaattcgcc
     8281 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     8341 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     8401 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     8461 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     8521 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     8581 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     8641 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     8701 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     8761 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     8821 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     8881 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     8941 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     9001 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     9061 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     9121 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     9181 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     9241 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     9301 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     9361 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     9421 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     9481 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     9541 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     9601 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     9661 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     9721 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     9781 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     9841 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     9901 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     9961 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
    10021 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
    10081 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
    10141 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
    10201 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
    10261 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
    10321 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
    10381 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
    10441 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
    10501 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
    10561 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
    10621 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
    10681 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
    10741 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
    10801 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
    10861 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
    10921 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
    10981 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
    11041 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
    11101 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct
    11161 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc
    11221 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac
    11281 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat
    11341 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg
    11401 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
    11461 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
    11521 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
    11581 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
    11641 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
    11701 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
    11761 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
    11821 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
    11881 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
    11941 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
    12001 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
    12061 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
    12121 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
    12181 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
    12241 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
    12301 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
    12361 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
    12421 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
    12481 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
    12541 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
    12601 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata
    12661 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat
    12721 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag
    12781 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga
    12841 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat
    12901 ccattcgatt agtgaacgga tctcgacggt atcggttaac
//
