LOCUS       sequence-497371-       13715 bp    DNA     circular SYN 15-JAN-2026
DEFINITION  Expresses Cas9 and mNeonGreen. Barcode is added to the vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13715)
  AUTHORS   Teh SSK, Halper-Stromberg E, Morsberger L, Bennett A, Bowland K, 
            Skaist A, Cai F, Liang H, Hruban RH, Roberts NJ, Scharpf RB, Zou YS,
            Eshleman JR
  TITLE     Mechanism of delayed cell death following simultaneous CRISPR-Cas9 
            targeting in pancreatic cancers.
  JOURNAL   bioRxiv [Preprint]. 2023 Apr 5:2023.04.03.535384. doi: 
            10.1101/2023.04.03.535384.
  PUBMED    37066222
REFERENCE   2  (bases 1 to 13715)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 15, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2023 Apr 5:2023.04.03.535384. doi: 
            10.1101/2023.04.03.535384."
COMMENT     Sequence Label: pLenti-Cas9-T2A-mNeonGreen-2-bc6
FEATURES             Location/Qualifiers
     source          1..13715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..57
                     /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             58..456
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    481..1069
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(534..554)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(952..963)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1606..1786
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(1812..1829)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1818..2042
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2088..2516
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2175..2194)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2385..2406
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2525..2545)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2690..2824
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2751..2770
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2886..2933
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2952..3326
                     /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    3456..3577
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3493..3512)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3547..3566
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3626..3642)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3626..3642)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3639..3661)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3650..3666
                     /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(3674..3680,3681..3698,3699..3704))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3719..3740
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3857..3874)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4028..4616)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4108..4127)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4787..5578,5579..5647))
                     /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     5410..5429
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5648..5752)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5827..5846)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6018..6397
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6399..6597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6552..6572
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6615..6795
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6842..6967
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7460..7693
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7878..7922
                     /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    8220..8337
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8430..8641
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      8649..8658
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             8658..8723
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             8730..8750
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8775..12875
                     /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             8775..12875
                     /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             12876..12923
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             12930..12983
                     /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             12984..13688
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      13236..13245
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc tggaccgatg
       61 gccaagcctt tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac
      121 agcatcccca tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc
      181 atcttcactg gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg
      241 ctgggcactg ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat
      301 gagaacaggg gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat
      361 cctgggatca aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt
      421 gaattgctgc cctctggtta tgtgtgggag ggctaagaat tcgatatcaa gcttatcggt
      481 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
      541 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
      601 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
      661 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
      721 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
      781 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
      841 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
      901 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
      961 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     1021 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcggtaaaac
     1081 gacggccaga attggcctcg agacctagaa aaacatggag caatcacaag tagcaataca
     1141 gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt
     1201 ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
     1261 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     1321 atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca
     1381 ccagggccag ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt
     1441 gagcaagaga aggtagaaga agccaatgaa ggagagaaca cccgcttgtt acaccctgtg
     1501 agcctgcatg ggatggatga cccggagaga gaagtattag agtggaggtt tgacagccgc
     1561 ctagcatttc atcacatggc ccgagagctg catccggact gtactgggtc tctctggtta
     1621 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     1681 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     1741 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagggc ccgtttaaac
     1801 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     1861 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     1921 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     1981 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2041 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     2101 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2161 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2221 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2281 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2341 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2401 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2461 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     2521 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     2581 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     2641 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     2701 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     2761 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     2821 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcag
     2881 cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga
     2941 ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg
     3001 ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg
     3061 acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg
     3121 tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg
     3181 agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga
     3241 tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc
     3301 acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct
     3361 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     3421 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     3481 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3541 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     3601 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3661 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3721 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3781 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3841 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3901 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3961 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4021 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4081 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4141 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4201 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4261 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4321 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4381 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4441 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4501 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4561 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4621 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4681 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4741 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4801 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4861 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4921 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4981 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5041 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5101 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5161 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5221 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5281 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5341 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5401 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5461 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5521 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5581 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5641 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5701 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5761 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
     5821 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     5881 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     5941 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     6001 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
     6061 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     6121 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     6181 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     6241 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     6301 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     6361 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     6421 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     6481 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     6541 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagcgc
     6601 gttttgcctg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
     6661 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     6721 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     6781 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
     6841 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
     6901 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
     6961 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
     7021 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
     7081 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
     7141 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
     7201 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
     7261 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca
     7321 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
     7381 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
     7441 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
     7501 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
     7561 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
     7621 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
     7681 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
     7741 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
     7801 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
     7861 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
     7921 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag
     7981 taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta
     8041 ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca
     8101 ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag
     8161 tgaacggatc ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt catccacaat
     8221 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata
     8281 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg
     8341 gtttattaca gggacagcag agatccagtt tggttaatta gctagctagg tcttgaaagg
     8401 agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
     8461 gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc gcggggtaaa
     8521 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
     8581 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
     8641 ggaccggtgc caccatggac tataaggacc acgacggaga ctacaaggat catgatattg
     8701 attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg
     8761 gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc aactctgtgg
     8821 gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca
     8881 acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg
     8941 aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga
     9001 accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct
     9061 tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc
     9121 ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc
     9181 acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg
     9241 ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg
     9301 acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg
     9361 aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga
     9421 gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc
     9481 tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg
     9541 acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca
     9601 acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt
     9661 ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc
     9721 tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag
     9781 ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga
     9841 acggctacgc cggctacatt gacggcggag ccagccagga agagttctac aagttcatca
     9901 agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg
     9961 acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg
    10021 gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc
    10081 gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca
    10141 ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga
    10201 acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca
    10261 acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt
    10321 acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc
    10381 ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc
    10441 ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact
    10501 ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc
    10561 tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg
    10621 aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga
    10681 aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca
    10741 ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca
    10801 agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga
    10861 tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg
    10921 gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca
    10981 tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg
    11041 agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca
    11101 gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga
    11161 aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc
    11221 agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg
    11281 atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc
    11341 tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga
    11401 agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt
    11461 tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca
    11521 tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact
    11581 cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca
    11641 ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg
    11701 agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc
    11761 tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg
    11821 acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact
    11881 tcttctacag caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga
    11941 tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg
    12001 gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa
    12061 agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg
    12121 ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc
    12181 ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac
    12241 tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga
    12301 atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca
    12361 agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg
    12421 ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt
    12481 acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc
    12541 tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct
    12601 ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc
    12661 accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca
    12721 atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca
    12781 ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg
    12841 agacacggat cgacctgtct cagctgggag gcgacaaaag gccggcggcc acgaaaaagg
    12901 ccggccaggc aaaaaagaaa aaggaattcg agggcagagg aagtctgcta acatgcggtg
    12961 acgtcgagga gaatcctggc ccagtgagca agggcgagga ggataacatg gcctctctcc
    13021 cagcgacaca tgagttacac atctttggct ccatcaacgg tgtggacttt gacatggtgg
    13081 gtcagggcac cggcaatcca aatgatggtt atgaggagtt aaacctgaag tccaccaagg
    13141 gtgacctcca gttctccccc tggattctgg tccctcatat cgggtatggc ttccatcagt
    13201 acctgcccta ccctgacggg atgtcgcctt tccaggccgc catggtagat ggctccggat
    13261 accaagtcca tcgcacaatg cagtttgaag atggtgcctc ccttactgtt aactaccgct
    13321 acacctacga gggaagccac atcaaaggag aggcccaggt gaaggggact ggtttccctg
    13381 ctgacggtcc tgtgatgacc aactcgctga ccgctgcgga ctggtgcagg tcgaagaaga
    13441 cttaccccaa cgacaaaacc atcatcagta cctttaagtg gagttacacc actggaaatg
    13501 gcaagcgcta ccggagcact gcgcggacca cctacacctt tgccaagcca atggcggcta
    13561 actatctgaa gaaccagccg atgtacgtgt tccgtaagac ggagctcaag cactccaaga
    13621 ccgagctcaa cttcaaggag tggcaaaagg cctttaccga tgtgatgggc atggacgagc
    13681 tgtacaagtc tagaagcgct ggtaccggat ccggc
//
