LOCUS       sequence-416265-       12878 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid express EFS promoter driven human codon optimized 
            SpdCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12878)
  AUTHORS   Mahata B, Cabrera A, Brenner DA, Guerra-Resendez RS, Li J, Goell J, 
            Wang K, Guo Y, Escobar M, Parthasarathy AK, Szadowski H, Bedford G, 
            Reed DR, Kim S, Hilton IB
  TITLE     Compact engineered human mechanosensitive transactivation modules 
            enable potent and versatile synthetic transcriptional control.
  JOURNAL   Nat Methods. 2023 Oct 9. doi: 10.1038/s41592-023-02036-1.
  PUBMED    37813990
REFERENCE   2  (bases 1 to 12878)
  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 
            Methods. 2023 Oct 9. doi: 10.1038/s41592-023-02036-1."
COMMENT     Sequence Label: EFS-SpdCas9
FEATURES             Location/Qualifiers
     source          1..12878
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(344..355)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             533..766
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(792..809)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    798..1022
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1068..1496
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1155..1174)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1365..1386
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1505..1525)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1510..1839
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1690..1825
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1752..1771
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        1887..1934
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1953..2327
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    2457..2578
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2494..2513)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2548..2567
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2627..2643)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2627..2643)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2640..2662)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2651..2667
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2675..2681,2682..2699,2700..2705))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2720..2741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2858..2875)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3029..3617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3109..3128)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3788..4579,4580..4648))
                     /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     4411..4430
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4649..4753)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4828..4847)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5019..5398
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5400..5598
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5553..5573
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5616..5796
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5843..5968
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6461..6694
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6879..6923
                     /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    7221..7338
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        7470..7545
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        7607..7818
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7837..7846
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7843..7869,7870..7872,7873..10359,10360..10362,
                     10363..11946)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7843..11946
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             11947..11994
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             12031..12051
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             12052..12075
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             12082..12138
                     /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             12139..12735
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     12632..12652
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    join(12751..12878,1..461)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(12804..12824)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
ORIGIN
        1 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
       61 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
      121 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
      181 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
      241 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
      301 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
      361 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
      421 ccctcagacg agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg
      481 acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg
      541 ctaattcact cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta
      601 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
      661 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
      721 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagggc ccgtttaaac
      781 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
      841 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
      901 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
      961 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     1021 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     1081 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1141 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     1201 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1261 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     1321 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     1381 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     1441 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     1501 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     1561 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     1621 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     1681 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     1741 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     1801 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     1861 tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca
     1921 tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg
     1981 tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct
     2041 cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca
     2101 tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg
     2161 gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct
     2221 ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg
     2281 acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtgctacgag
     2341 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     2401 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     2461 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     2521 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     2581 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     2641 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     2701 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     2761 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     2821 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     2881 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     2941 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3001 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3061 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3121 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3181 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3241 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3301 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3361 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3421 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     3481 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3541 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3601 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3661 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3721 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3781 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3841 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     3901 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3961 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4021 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4081 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4141 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4201 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4261 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4321 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4381 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4441 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4501 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4561 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     4621 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     4681 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     4741 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     4801 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4861 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     4921 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     4981 tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta
     5041 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     5101 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     5161 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     5221 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     5281 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     5341 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     5401 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     5461 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     5521 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     5581 ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct
     5641 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     5701 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     5761 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
     5821 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
     5881 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
     5941 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     6001 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     6061 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     6121 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     6181 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     6241 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     6301 gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg
     6361 aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa
     6421 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt
     6481 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca
     6541 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc
     6601 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg
     6661 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac
     6721 tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga
     6781 tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa
     6841 tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg
     6901 aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata
     6961 taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac
     7021 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc
     7081 caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca
     7141 gagacagatc cattcgatta gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa
     7201 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     7261 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     7321 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt ttggttaatt
     7381 aaggtaccga tatcactagt gttccattaa ctgcgctaaa aataattttt aaatcttttt
     7441 tagcttcttg ctcttttttg tacgtctctg ttttagagct agaaatagca agttaaaata
     7501 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgaattcgct
     7561 agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca
     7621 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg tgcctagaga
     7681 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     7741 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     7801 tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa gaagtacagc
     7861 atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag
     7921 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac
     7981 ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga
     8041 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc
     8101 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg
     8161 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg
     8221 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc
     8281 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc
     8341 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc
     8401 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg
     8461 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc
     8521 gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg
     8581 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg
     8641 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac
     8701 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg
     8761 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac
     8821 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag
     8881 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga
     8941 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc
     9001 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac
     9061 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag
     9121 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc
     9181 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc
     9241 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct
     9301 tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag
     9361 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa
     9421 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag
     9481 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag
     9541 gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg
     9601 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc
     9661 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt
     9721 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa
     9781 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg
     9841 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac
     9901 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag
     9961 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat
    10021 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag
    10081 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag
    10141 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag
    10201 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg
    10261 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag
    10321 gaactggaca tcaaccggct gtccgactac gatgtggacg ccatcgtgcc tcagagcttt
    10381 ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag
    10441 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg
    10501 ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc
    10561 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag
    10621 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat
    10681 gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc
    10741 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac
    10801 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc
    10861 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag
    10921 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc
    10981 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac
    11041 ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg
    11101 ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc
    11161 aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg
    11221 gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg
    11281 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc
    11341 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc
    11401 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg
    11461 gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg
    11521 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag
    11581 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg
    11641 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat
    11701 ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc
    11761 gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac
    11821 tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc
    11881 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga
    11941 ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa gaaaagtggt
    12001 ggaggaagtg gcgggtcagg gtcgggatcc ccgaaaaaga aacgcaaagt tgattacaaa
    12061 gacgatgacg ataagtgagg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc
    12121 gaagagaatc ctggaccgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
    12181 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
    12241 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
    12301 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
    12361 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
    12421 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
    12481 caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag
    12541 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
    12601 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
    12661 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
    12721 aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc tggattacaa aatttgtgaa
    12781 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
    12841 atgcctttgt atcatgctat tgcttcccgt atggcttt
//
