LOCUS       sequence-191832-       13668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  single-vector system with EGFP reporter for use in CROPseq knockout 
            screens.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13668)
  AUTHORS   Hewitt
  TITLE     CROPseq-Guide-EFS-SpCas9-P2A-EGFP
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 13668)
  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: 
            "Unpublished"
COMMENT     Sequence Label: CROPseq-Guide-EFS-SpCas9-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..13668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     36..56
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        38..56
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        84..310
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             311..491
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    538..663
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1156..1389
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1574..1618
                     /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    1799..1916
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2105..2316
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2335..2344
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2341..6444
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2341..6444
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6445..6492
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6493..6516
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6526..6582
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(6583..6585,6586..7299)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6886..6905)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7233..7254
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    7315..7903
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7368..7388)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7786..7797)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        8059..8307
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8059..8079
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8230..8249
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        10189..10264
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             10317..10497
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    10575..10696
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10612..10631)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10666..10685
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      10736..10871
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10891..10910)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10892..10910)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10920..10937)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10920..10936)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10929..10951)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11078..11533
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11165..11184)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11375..11396
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11559..11663
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11664..11732,11733..12524)
                     /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(11882..11901)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12695..13283
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13184..13203
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13437..13454
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    13571..13592
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13607..13612,13613..13630,13631..13637)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13645..13661
                     /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     join(13650..13668,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
       61 aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat
      121 ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt
      181 ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg
      241 gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg
      301 atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
      361 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
      421 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
      481 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
      541 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
      601 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
      661 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
      721 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
      781 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
      841 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
      901 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
      961 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc
     1021 tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa
     1081 aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa
     1141 aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat
     1201 gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca
     1261 gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt
     1321 ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca
     1381 acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg
     1441 gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga
     1501 gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca
     1561 aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg
     1621 gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg
     1681 aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca
     1741 gggatattca ccattatcgt ttcagaatct tgagacaaat ggcagtattc atccacaatt
     1801 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
     1861 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
     1921 tttattacag ggacagcaga gatccacttt ggcgccggct cgagggggcc cgggtgcaaa
     1981 gatggataaa gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga
     2041 aaggagtggg aattggctcc gtaggtcttg aaaggagtgg gaattggctc cggtgcccgt
     2101 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
     2161 tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
     2221 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
     2281 gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag cgctgccacc
     2341 atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg
     2401 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
     2461 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
     2521 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
     2581 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
     2641 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
     2701 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
     2761 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
     2821 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     2881 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     2941 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     3001 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac
     3061 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     3121 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     3181 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     3241 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     3301 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     3361 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     3421 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     3481 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     3541 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     3601 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     3661 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     3721 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     3781 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     3841 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     3901 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     3961 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     4021 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     4081 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     4141 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     4201 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     4261 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     4321 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     4381 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     4441 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     4501 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     4561 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     4621 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     4681 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     4741 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     4801 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat
     4861 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     4921 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     4981 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     5041 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     5101 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     5161 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     5221 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     5281 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     5341 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     5401 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     5461 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     5521 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     5581 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     5641 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     5701 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     5761 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     5821 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     5881 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     5941 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     6001 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     6061 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     6121 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     6181 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     6241 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     6301 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     6361 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     6421 gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc tggacaggct
     6481 aagaagaaga aagattacaa agacgatgac gataagggat ccggcgcaac aaacttctct
     6541 ctgctgaaac aagccggaga tgtcgaagag aatcctggac cggtgagcaa gggcgaggag
     6601 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     6661 ttcagcgtgt ctggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     6721 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     6781 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     6841 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     6901 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     6961 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     7021 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggc gaacttcaag
     7081 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     7141 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     7201 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     7261 gccgggatca ctctcggcat ggacgagctg tacaagtaaa cgcgttaagt cgacaatcaa
     7321 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     7381 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     7441 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     7501 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     7561 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     7621 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     7681 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     7741 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     7801 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     7861 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa
     7921 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     7981 ggctaattca ctcccaacga agacaagatt tcaccattat cgtttcagac ccacctccca
     8041 accccgaggg gacccagaga gggcctattt cccatgattc cttcatattt gcatatacga
     8101 tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta
     8161 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     8221 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     8281 tatatcttgt ggaaaggacg aaacaccgga gacggttgta aatgagcaca caaaatacac
     8341 atgctaaaat attatattct atgaccttta taaaatcaac caaaatcttc tttttaataa
     8401 ctttagtatc aataattaga atttttatgt tcctttttgc aaacttttaa taaaaatgag
     8461 caaaataaaa aaacgctagt tttagtaact cgcgttgttt tcttcacctt taataatagc
     8521 tactccacca cttgttccta agcggtcagc tcctgcttca atcatttttt gagcatcttc
     8581 aaatgttcta actccaccag ctgctttaac taaagcattg tctttaacaa ctgacttcat
     8641 tagtttaaca tcttcaaatg ttgcacctga ttttgaaaat cctgttgatg ttttaacaaa
     8701 ttctaatcca gcttcaacag ctatttcaca agctttcatg atttcttctt ttgttaataa
     8761 acaattttcc ataatacatt taacaacatg tgatccagct gcttttttta cagctttcat
     8821 gtcttctaaa actaattcat aatttttgtc ttttaatgca ccaatattta ataccatatc
     8881 aatttctgtt gcaccatctt taattgcttc agaaacttcg aatgcttttg tagctgttgt
     8941 gcatgcacct agaggaaaac ctacaacatt tgttattcct acatttgtgc cttttaataa
     9001 ttctttacaa tagcttgttc aatatgaatt aacacaaact gttgcaaaat caaattcaat
     9061 tgcttcatca cataattgtt taatttcagc tttcgtagca tcttgtttta ataatgtgtg
     9121 atctatatat ttgtttagtt tcattttttc tcctatatat tcatttttaa ttttaattct
     9181 ttaataattt cgtctacttt aactttagcg ttttgaacag attcaccaac acctataaaa
     9241 taaattttta gtttaggttc agttccactt gggcgaacag caaatcatga cttatcttct
     9301 aaataaaatt ttagtaagtc ttgtcctggc atattataca ttccatcgat gtagtcttca
     9361 acattaacaa ctttaagtcc agcaatttga gttaagggtg ttgctctcaa tgatttcatt
     9421 aatggttcaa tttttaattt cttttcttct ggtttaaaat tcaagtttaa agtgaaagtg
     9481 taatatgcac ccatttcttt aaataaatct tctaaatagt ctactaatgt tttattttgt
     9541 tttttataaa atcaagcagc ctctgctatt aatatagaag cttgtattcc atctttatct
     9601 ctagctgagt catcaattac atatccataa ctttcttcat aagcaaaaac aaaatttaat
     9661 ccgttatctt cttctttagc aatttctcta cccattcatt taaatccagt taaagttttt
     9721 acaatattaa ctccatattt ttcatgagcg attctatcac ccaaatcact tgttacaaaa
     9781 cttgaatata gagccggatt ttttggaatg ctatttaagc gttttagatt tgataatttt
     9841 caatcaatta aaattggtcc tgtttgattt ccatctaatc ttacaaaatg accatcatgt
     9901 tttattgcca ttccaaatct gtcagcatct gggtcattca taataataat atctgcatca
     9961 tgtttaatac catattcaag cggtattttt catgcaggat caaattctgg atttggattt
    10021 acaacatttt taaatgtttc atcttcaaat gcatgctctt caacctcaat aacgttatat
    10081 cctgattcac gtaatatttt tggggtaaat ttagttcctg ttccattaac tgcgctaaaa
    10141 ataattttta aatctttttt agcttcttgc tcttttttgt acgtctctgt tttagagcta
    10201 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
    10261 gtgctttttt aagcttggcg taactagatc ttgagacact gctttttgct tgtactgggt
    10321 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
    10381 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
    10441 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
    10501 cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
    10561 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
    10621 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
    10681 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc
    10741 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
    10801 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
    10861 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
    10921 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
    10981 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
    11041 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
    11101 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
    11161 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
    11221 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
    11281 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
    11341 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
    11401 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
    11461 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
    11521 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
    11581 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
    11641 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
    11701 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
    11761 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
    11821 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
    11881 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
    11941 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
    12001 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
    12061 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
    12121 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
    12181 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
    12241 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
    12301 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
    12361 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
    12421 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
    12481 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
    12541 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
    12601 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
    12661 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
    12721 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
    12781 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
    12841 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
    12901 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
    12961 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
    13021 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
    13081 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
    13141 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
    13201 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
    13261 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
    13321 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
    13381 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
    13441 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
    13501 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
    13561 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
    13621 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcaca
//
