LOCUS       sequence-428814-       13739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression vector for an inducible Cas9-P2A-Puromycin 
            resistance casette with two empty sgRNA cloning sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13739)
  TITLE     Selectable lentivectors delivering dual gRNAs and destabilized Cas9
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 13739)
  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            "Unpublished"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: pDual_dsCas9_Puro
FEATURES             Location/Qualifiers
     source          1..13739
                     /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        2608..2848
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     misc_RNA        2860..2935
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3026..3266
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     misc_RNA        3274..3349
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3411..3622
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3636..3645
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3642..3644,3645..3965)
                     /codon_start=1
                     /product="destabilization domain that can be stabilized by 
                     Shield1 in the ProteoTuner(TM) system"
                     /label=DD
                     /note="L106P mutant of FKBP12"
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKPE"
     CDS             3975..8078
                     /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             3975..8078
                     /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             8079..8126
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8127..8150
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             8160..8216
                     /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             8220..8816
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(8220..8239)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8716..8736
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8831..9419
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8884..8904)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9302..9313)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9491..9724
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9750..9767)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9756..9980
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10026..10454
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10113..10132)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10323..10344
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10463..10483)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10468..10797
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10648..10783
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10710..10729
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10845..10892
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10911..11285
                     /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    11415..11536
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11452..11471)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11506..11525
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11585..11601)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11585..11601)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11598..11620)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11609..11625
                     /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(11633..11639,11640..11657,11658..11663))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11678..11699
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11816..11833)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11987..12575)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12067..12086)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12746..13537,13538..13606))
                     /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     13369..13388
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13607..13711)
                     /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 aggtaccgag ggcctatttc ccatgattcc ttcatatttg
     2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2821 ttggctttat atatcttgtg gaaaggacga aacctgcagg ttttagagct agaaatagca
     2881 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     2941 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc
     3001 aattctgcag acaaatggct ctagagaggg cctatttccc atgattcctt catatttgca
     3061 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     3121 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     3181 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     3241 ggctttatat atcttgtgga aaggaccgtc tctgttttag agctagaaat agcaagttaa
     3301 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgaatt
     3361 cgctagctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt gggcagagcg
     3421 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat ccggtgccta
     3481 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
     3541 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
     3601 cgggtttgcc gccagaacac aggaccggtt ctagagccac catgggagtg caggtggaaa
     3661 ccatctcccc aggtgacggg cgcaccttcc ccaagcgcgg ccagacctgt gtggtgcact
     3721 acaccgggat gcttgaagat ggaaagaaag tcgattcctc ccgggacaga aacaagccct
     3781 ttaagtttat gctaggcaag caggaggtga tccgaggctg ggaagaaggg gttgcccaga
     3841 tgagtgtggg tcagagagcc aaactgacta tatctccaga ttatgcctat ggtgccactg
     3901 ggcacccagg catcatccca ccacatgcca ctctcgtctt cgatgtggag cttctaaaac
     3961 cggaaccggc tagcatggac aagaagtaca gcatcggcct ggacatcggc accaactctg
     4021 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     4081 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg
     4141 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     4201 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     4261 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     4321 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     4381 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     4441 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     4501 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     4561 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     4621 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     4681 gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     4741 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     4801 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     4861 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     4921 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     4981 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     5041 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca
     5101 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     5161 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     5221 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     5281 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     5341 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     5401 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga
     5461 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     5521 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     5581 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca
     5641 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     5701 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     5761 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     5821 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     5881 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     5941 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     6001 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     6061 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     6121 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     6181 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     6241 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     6301 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     6361 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     6421 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     6481 acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg
     6541 tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg
     6601 tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa
     6661 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     6721 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
     6781 actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga
     6841 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     6901 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
     6961 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
     7021 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
     7081 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
     7141 agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata
     7201 agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga
     7261 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
     7321 gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
     7381 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
     7441 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga
     7501 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
     7561 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
     7621 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
     7681 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
     7741 agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt
     7801 tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca
     7861 agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga
     7921 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
     7981 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
     8041 acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga
     8101 aggctggaca ggctaagaag aagaaagatt acaaagacga tgacgataag ggatccggag
     8161 ctactaactt cagcctgctg aagcaggctg gggacgtgga ggagaaccct ggacctagga
     8221 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     8281 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     8341 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     8401 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     8461 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     8521 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     8581 ccgcgtggtt cctggccacc gtcggagtga gccccgacca ccagggcaag ggtctgggca
     8641 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     8701 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     8761 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgccgatc
     8821 ctaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     8881 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     8941 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     9001 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     9061 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     9121 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     9181 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     9241 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     9301 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     9361 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg
     9421 tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     9481 aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt
     9541 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     9601 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     9661 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     9721 agcagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     9781 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     9841 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     9901 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     9961 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
    10021 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
    10081 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
    10141 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
    10201 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
    10261 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
    10321 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
    10381 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
    10441 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
    10501 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
    10561 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
    10621 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
    10681 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
    10741 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
    10801 ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat
    10861 cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt
    10921 tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga
    10981 ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg
    11041 acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg
    11101 cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca
    11161 cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc
    11221 gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg
    11281 actgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
    11341 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
    11401 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
    11461 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
    11521 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
    11581 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
    11641 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
    11701 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
    11761 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
    11821 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
    11881 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
    11941 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
    12001 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
    12061 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
    12121 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
    12181 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
    12241 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
    12301 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
    12361 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
    12421 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
    12481 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
    12541 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
    12601 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
    12661 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
    12721 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
    12781 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
    12841 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
    12901 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
    12961 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
    13021 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
    13081 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
    13141 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
    13201 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
    13261 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
    13321 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
    13381 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
    13441 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
    13501 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
    13561 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
    13621 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
    13681 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgac
//
