LOCUS       sequence-431403-       13520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockout, EF1a-driven Cas9 (Cas only).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13520)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 13520)
  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: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: pMV_AA088
FEATURES             Location/Qualifiers
     source          1..13520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2640..3818
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2871..3809
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3766..3786
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3823..3832
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3868..3888
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3895..7995
                     /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             3895..7995
                     /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             8002..8049
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8056..8136
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             8143..8196
                     /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             8203..8601
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    8612..9200
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8665..8685)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9083..9094)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9272..9505
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9531..9548)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9537..9761
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9807..10235
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9894..9913)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10104..10125
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10244..10264)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10249..10578
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10429..10564
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10491..10510
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10626..10673
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10692..11066
                     /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    11196..11317
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11233..11252)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11287..11306
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11366..11382)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11366..11382)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11379..11401)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11390..11406
                     /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(11414..11420,11421..11438,11439..11444))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11459..11480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11597..11614)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11768..12356)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11848..11867)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12527..13318,13319..13387))
                     /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     13150..13169
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13388..13492)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta aggtaccggg cagaggtctc gcttttttcg agacaacacg
     2641 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     2701 gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     2761 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     2821 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
     2881 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
     2941 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
     3001 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
     3061 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
     3121 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
     3181 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     3241 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     3301 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
     3361 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     3421 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     3481 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     3541 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     3601 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     3661 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     3721 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     3781 tggttcaaag tttttttctt ccatttcagg tgtcgtgaag gagccaccat ggccaaacgg
     3841 acagccgacg gaagcgagtt cgagtcacca aagaagaagc ggaaagtcgg cagcgacaag
     3901 aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac
     3961 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc
     4021 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg
     4081 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa
     4141 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag
     4201 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg
     4261 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg
     4321 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag
     4381 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag
     4441 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgct
     4501 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa
     4561 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggcaa cctgattgcc
     4621 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa
     4681 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc
     4741 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc
     4801 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag
     4861 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg
     4921 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt
     4981 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg
     5041 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg
     5101 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg
     5161 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc
     5221 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc
     5281 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac
     5341 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc
     5401 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag
     5461 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag
     5521 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag
     5581 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg
     5641 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac
     5701 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg
     5761 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc
     5821 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc
     5881 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg
     5941 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc
     6001 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac
     6061 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg
     6121 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg
     6181 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg
     6241 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc agtggaaaac
     6301 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac
     6361 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct
     6421 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac
     6481 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg
     6541 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc
     6601 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa
     6661 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac
     6721 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg
     6781 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac
     6841 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag
     6901 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc
     6961 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg
     7021 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc
     7081 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg
     7141 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc
     7201 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag
     7261 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg
     7321 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg
     7381 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa
     7441 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg
     7501 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga
     7561 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag
     7621 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag
     7681 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc
     7741 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga
     7801 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc
     7861 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg
     7921 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacgaat cgatttgtct
     7981 cagctgggag gcgatgggtc caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa
     8041 aagaaaaagg gatcctaccc atacgatgtt ccagattacg cttatcccta cgacgtgcct
     8101 gattatgcat acccatatga tgtccccgac tatgccggaa gcgagggcag aggaagcctt
     8161 ctaacatgcg gtgacgtgga ggagaatccc ggcccttccg ggatggccaa gcctttgtct
     8221 caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct
     8281 gaggactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc
     8341 aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct
     8401 gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc
     8461 ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc
     8521 atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct
     8581 ggttatgtgt gggagggcta ataaattcga aaatcaacct ctggattaca aaatttgtga
     8641 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     8701 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     8761 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     8821 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     8881 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     8941 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     9001 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     9061 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     9121 gctgccggct ctgcggcctc ttccgcgtcc tcgccttcgc cctcagacga gtcggatctc
     9181 cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat
     9241 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     9301 taagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     9361 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     9421 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     9481 tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc cgctgatcag cctcgactgt
     9541 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     9601 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     9661 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     9721 agataatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     9781 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     9841 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     9901 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     9961 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
    10021 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
    10081 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
    10141 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
    10201 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
    10261 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
    10321 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
    10381 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
    10441 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
    10501 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
    10561 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcag
    10621 cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga
    10681 ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg
    10741 ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg
    10801 acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg
    10861 tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg
    10921 agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga
    10981 tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc
    11041 acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct
    11101 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
    11161 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
    11221 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
    11281 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
    11341 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
    11401 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
    11461 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
    11521 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
    11581 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
    11641 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
    11701 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
    11761 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
    11821 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
    11881 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
    11941 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
    12001 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
    12061 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
    12121 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
    12181 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
    12241 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
    12301 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
    12361 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
    12421 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
    12481 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
    12541 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
    12601 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
    12661 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
    12721 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
    12781 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
    12841 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
    12901 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
    12961 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
    13021 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
    13081 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
    13141 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
    13201 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
    13261 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
    13321 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
    13381 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
    13441 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
    13501 cccgaaaagt gccacctgac
//
