LOCUS       sequence-236123-       14012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The plasmid codes for a Flag-spCas9 protein, a TagRFP-T fluorescent 
            protein and a puromycin resistance. The plasmid has two BsmBI 
            acceptor sites to insert gRNA expressing sequences..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14012)
  TITLE     Lenti spCas9 T2A iRFP670 P2A puro
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 14012)
  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            "Unpublished"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: pLenti spCas9 T2A TagRFP-T P2A puro
FEATURES             Location/Qualifiers
     source          1..14012
                     /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"
     CDS             complement(2594..2914)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATTVSY
                     LN"
     primer_bind     2764..2786
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2778..2795
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2779..2795
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2805..2824
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2805..2823
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2871..2891)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2871..2889)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2910..2926)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2934..2950
                     /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(2958..2964,2965..2982,2983..2988))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3003..3024
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3122..3333
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3341..3350
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3350..3415
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3422..3442
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3467..7567
                     /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             3467..7567
                     /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             7568..7615
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7622..7675
                     /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             7676..8407
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     CDS             8435..8491
                     /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             8492..9088
                     /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     8985..9005
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9104..9692
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9157..9177)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9575..9586)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9764..9997
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10023..10040)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10029..10253
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10299..10727
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10386..10405)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10596..10617
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10736..10756)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10741..11070
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10921..11056
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10983..11002
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11118..11165
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11184..11558
                     /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    11688..11809
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11725..11744)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11779..11798
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11858..11874)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    11882..11898
                     /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(11906..11912,11913..11930,11931..11936))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11951..11972
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12089..12106)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12260..12848)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12340..12359)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13019..13810,13811..13879))
                     /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     13642..13661
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13880..13984)
                     /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 agataagaga cggtggtggc gccgctacag ggcgcgtccc
     2641 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     2701 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     2761 tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat
     2821 agggcgaatt gggtaccggg ccccccctcg aggtcctcca gcttttgttc cctttagtga
     2881 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     2941 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     3001 taatgagtga gctaactcac attaattgcg ttgcgctcaa tgcccgcttt ccactgcatg
     3061 acgtctccac aatctagcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag
     3121 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     3181 tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     3241 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     3301 ctttttcgca acgggtttgc cgccagaaca caggaccggt gccaccatgg actataagga
     3361 ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg ataagatggc
     3421 cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccgaca agaagtacag
     3481 catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa
     3541 ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa
     3601 cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag
     3661 aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt
     3721 cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag agtccttcct
     3781 ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt
     3841 ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac
     3901 cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg
     3961 ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat
     4021 ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt
     4081 ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat
     4141 cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct
     4201 gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct
     4261 gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta
     4321 cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct
     4381 gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga
     4441 cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa
     4501 gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg
     4561 agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac
     4621 cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga
     4681 caacggcagc atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca
     4741 ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt
     4801 ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac
     4861 cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc
     4921 ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa
     4981 ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa
     5041 agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa
     5101 ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga
     5161 ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg
     5221 gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt
     5281 cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt
     5341 tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa
     5401 agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct
     5461 gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga
     5521 cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga
     5581 ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa
     5641 tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga
     5701 gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga
     5761 gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga
     5821 gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct
     5881 gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca
     5941 ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt
     6001 tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa
     6061 gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct
     6121 gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg
     6181 cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca
     6241 gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa
     6301 tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt
     6361 ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga
     6421 cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag
     6481 cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga
     6541 gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt
     6601 caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa
     6661 cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt
     6721 gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag
     6781 caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg
     6841 ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt
     6901 ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat
     6961 caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg
     7021 ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct
     7081 ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact
     7141 ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa
     7201 gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct
     7261 ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa
     7321 tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc
     7381 cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta
     7441 ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac
     7501 cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg
     7561 aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaaggaatt
     7621 cgagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaatggt
     7681 gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctataca tggagggcac
     7741 cgtgaacaac caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     7801 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
     7861 ggctaccagc ttcatgtacg gcagcagaac cttcatcaac cacacccagg gcatccccga
     7921 tttctttaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga
     7981 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
     8041 cgtcaagatc agaggggtga acttcccatc caacggccct gtgatgcaga agaaaacact
     8101 cggctgggag gccaacaccg agatgctgta ccccgctgac ggcggcctgg aaggcagaac
     8161 cgacatggcc ctgaagctcg tgggcggggg ccacctgatc tgcaacttca agaccacata
     8221 cagatccaag aaacccgcta agaacctcaa gatgcccggc gtctactatg tggaccacag
     8281 actggaaaga atcaaggagg ccgacaaaga gacctacgtc gagcagcacg aggtggctgt
     8341 ggccagatac tgcgacctcc ctagcaaact ggggcacaaa cttaatggca tggacgagct
     8401 gtacaagtct agaagcgctg gtaccggatc cggcgcaaca aacttctctc tgctgaaaca
     8461 agccggagat gtcgaagaga atcctggacc gaccgagtac aagcccacgg tgcgcctcgc
     8521 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     8581 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     8641 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     8701 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     8761 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     8821 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc
     8881 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     8941 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     9001 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     9061 gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac ctctggatta
     9121 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     9181 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     9241 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     9301 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     9361 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     9421 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     9481 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     9541 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     9601 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     9661 gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag
     9721 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     9781 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
     9841 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     9901 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     9961 ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa cccgctgatc
    10021 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
    10081 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
    10141 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
    10201 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
    10261 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
    10321 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
    10381 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
    10441 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
    10501 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
    10561 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
    10621 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
    10681 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
    10741 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
    10801 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
    10861 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
    10921 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
    10981 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
    11041 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
    11101 ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat
    11161 acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg
    11221 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
    11281 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
    11341 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
    11401 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
    11461 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
    11521 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt
    11581 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
    11641 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
    11701 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
    11761 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
    11821 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
    11881 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
    11941 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
    12001 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
    12061 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
    12121 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
    12181 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
    12241 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
    12301 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
    12361 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
    12421 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
    12481 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
    12541 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
    12601 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
    12661 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
    12721 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
    12781 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
    12841 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
    12901 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
    12961 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
    13021 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
    13081 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
    13141 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
    13201 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
    13261 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
    13321 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
    13381 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
    13441 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
    13501 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
    13561 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
    13621 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
    13681 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
    13741 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
    13801 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
    13861 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
    13921 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
    13981 cgcgcacatt tccccgaaaa gtgccacctg ac
//
