LOCUS       sequence-333283-       14507 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid allows efficient KO of the firefly luciferase gene, 
            while expressing the far red fluorescent protein iRFP670 and 
            puromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14507)
  AUTHORS   Deffieu MS, Clement CMH, Dorobantu CM, Partiot E, Bare Y, Faklaris 
            O, Riviere B, Ayala-Nunez NV, Baumert TF, Ronde P, Mely Y, Lucansky 
            V, Gaudin R
  TITLE     Occludin stalls HCV particle dynamics apart from hepatocyte tight 
            junctions, promoting virion internalization.
  JOURNAL   Hepatology. 2022 Apr 7. doi: 10.1002/hep.32514.
  PUBMED    35388524
REFERENCE   2  (bases 1 to 14507)
  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: "Hepatology.
            2022 Apr 7. doi: 10.1002/hep.32514."
COMMENT     Sequence Label: pLenti KO Luc Firefly 2xgRNA - spCas9 iRFP670 ...
FEATURES             Location/Qualifiers
     source          1..14507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..33)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        18..347
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      198..333
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     260..279
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        395..442
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             461..835
                     /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    965..1086
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1002..1021)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1056..1075
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1135..1151)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1135..1151)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1148..1170)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1159..1175
                     /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(1183..1189,1190..1207,1208..1213))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1228..1249
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1366..1383)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1537..2125)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1617..1636)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2296..3087,3088..3156))
                     /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     2919..2938
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3157..3261)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3336..3355)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3527..3906
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3908..4106
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4061..4081
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4124..4304
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4351..4476
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4969..5202
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5387..5431
                     /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    5729..5846
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5910..6150
                     /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."
     primer_bind     5910..5930
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6081..6100
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6173..6248
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6262..6476
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     6397..6416
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        6497..6572
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6633..6844
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6852..6861
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6861..6926
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6933..6953
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6978..11078
                     /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             6978..11078
                     /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             11079..11126
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             11133..11186
                     /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             11187..11237
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     regulatory      11253..11262
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             11259..12191
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="MARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAG
                     AFFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLH
                     RHDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVM
                     LYRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSR
                     IVPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYE
                     PRAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     CDS             12219..12275
                     /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             12276..12872
                     /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     12769..12789
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    12888..13476
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(12941..12961)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(13359..13370)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             13548..13781
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(13807..13824)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    13813..14037
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(14083..14507,1..4)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(14170..14189)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     14380..14401
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
       61 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
      121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
      181 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
      241 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
      301 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
      361 cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta
      421 tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc
      481 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct
      541 cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac
      601 cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg
      661 ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg
      721 ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc
      781 cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt
      841 gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
      901 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
      961 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1021 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     1081 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     1141 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     1201 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     1261 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1321 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     1381 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     1441 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     1501 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     1561 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1621 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1681 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     1741 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     1801 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     1861 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     1921 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     1981 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2041 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2101 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2161 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2221 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2281 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     2341 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     2401 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     2461 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     2521 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     2581 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     2641 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     2701 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     2761 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     2821 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     2881 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     2941 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3001 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3061 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3121 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     3181 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3241 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     3301 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3361 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     3421 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     3481 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
     3541 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     3601 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     3661 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     3721 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     3781 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     3841 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     3901 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     3961 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     4021 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     4081 gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc tctggttaga
     4141 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     4201 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     4261 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
     4321 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
     4381 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
     4441 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
     4501 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
     4561 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
     4621 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     4681 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     4741 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     4801 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     4861 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     4921 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     4981 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     5041 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     5101 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     5161 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     5221 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     5281 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     5341 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     5401 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     5461 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     5521 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     5581 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
     5641 gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct
     5701 gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca
     5761 gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa
     5821 aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt
     5881 ggttaattaa gataaaggtc gggcaggaag agggcctatt tcccatgatt ccttcatatt
     5941 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     6001 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     6061 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     6121 tcttggcttt atatatcttg tggaaaggac ggcttcgaaa tgtccgttcg gtgttttaga
     6181 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     6241 gtcggtgctt tttttctcga ggaacgctga cgtcatcaac ccgctccaag gaatcgcggg
     6301 cccagtgtca ctaggcggga acacccagcg cgcgtgcgcc ctggcaggaa gatggctgtg
     6361 agggacaggg gagtggcgcc ctgcaatatt tgcatgtcgc tatgtgttct gggaaatcac
     6421 cataaacgtg aaatgtcttt ggatttggga atcttataag ttctgtatga gaccaccccg
     6481 gcgccattct atccgcgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     6541 atcaacttga aaaagtggca ccgagtcggt gcttttttta caatctagct aggtcttgaa
     6601 aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     6661 cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt ggcgcggggt
     6721 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     6781 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     6841 acaggaccgg tgccaccatg gactataagg accacgacgg agactacaag gatcatgata
     6901 ttgattacaa agacgatgac gataagatgg ccccaaagaa gaagcggaag gtcggtatcc
     6961 acggagtccc agcagccgac aagaagtaca gcatcggcct ggacatcggc accaactctg
     7021 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     7081 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg
     7141 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     7201 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     7261 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     7321 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     7381 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     7441 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     7501 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     7561 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     7621 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     7681 gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     7741 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     7801 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     7861 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     7921 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     7981 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     8041 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca
     8101 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     8161 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     8221 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     8281 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     8341 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     8401 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga
     8461 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     8521 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     8581 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca
     8641 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     8701 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     8761 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     8821 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     8881 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     8941 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     9001 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     9061 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     9121 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     9181 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     9241 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     9301 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     9361 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     9421 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     9481 acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg
     9541 tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg
     9601 tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa
     9661 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     9721 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
     9781 actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga
     9841 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     9901 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
     9961 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
    10021 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
    10081 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
    10141 agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata
    10201 agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga
    10261 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
    10321 gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
    10381 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
    10441 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga
    10501 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
    10561 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
    10621 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
    10681 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
    10741 agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt
    10801 tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca
    10861 agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga
    10921 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
    10981 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
    11041 acgagacacg gatcgacctg tctcagctgg gaggcgacaa aaggccggcg gccacgaaaa
    11101 aggccggcca ggcaaaaaag aaaaaggaat tcgagggcag aggaagtctg ctaacatgcg
    11161 gtgacgtcga ggagaatcct ggcccaatgg gcgtggccga cttgatcaag aagttcgagt
    11221 ccatctccaa ggaggagggg gatccaccgg tcgccaccat ggcgcgtaag gtcgatctca
    11281 cctcctgcga tcgcgagccg atccacatcc ccggcagcat tcagccgtgc ggctgcctgc
    11341 tagcctgcga cgcgcaggcg gtgcggatca cgcgcattac ggaaaatgcc ggcgcgttct
    11401 ttggacgcga aactccgcgg gtcggtgagc tactcgccga ttacttcggc gagaccgaag
    11461 cccatgcgct gcgcaacgca ctggcgcagt cctccgatcc aaagcgaccg gcgctgatct
    11521 tcggttggcg cgacggcctg accggccgca ccttcgacat ctcactgcat cgccatgacg
    11581 gtacatcgat catcgagttc gagcctgcgg cggccgaaca ggccgacaat ccgctgcggc
    11641 tgacgcggca gatcatcgcg cgcaccaaag aactgaagtc gctcgaagag atggccgcac
    11701 gggtgccgcg ctatctgcag gcgatgctcg gctatcaccg cgtgatgttg taccgcttcg
    11761 cggacgacgg ctccgggatg gtgatcggcg aggcgaagcg cagcgacctc gagagctttc
    11821 tcggtcagca ctttccggcg tcgctggtcc cgcagcaggc gcggctactg tacttgaaga
    11881 acgcgatccg cgtggtctcg gattcgcgcg gcatcagcag ccggatcgtg cccgagcacg
    11941 acgcctccgg cgccgcgctc gatctgtcgt tcgcgcacct gcgcagcatc tcgccctgcc
    12001 atctcgaatt tctgcggaac atgggcgtca gcgcctcgat gtcgctgtcg atcatcattg
    12061 acggcacgct atggggattg atcatctgtc atcattacga gccgcgtgcc gtgccgatgg
    12121 cgcagcgcgt cgcggccgaa atgttcgccg acttcttatc gctgcacttc accgccgccc
    12181 accaccaacg ctctagaagc gctggtaccg gatccggcgc aacaaacttc tctctgctga
    12241 aacaagccgg agatgtcgaa gagaatcctg gaccgaccga gtacaagccc acggtgcgcc
    12301 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
    12361 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
    12421 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
    12481 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
    12541 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
    12601 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag
    12661 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
    12721 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
    12781 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
    12841 cctggtgcat gacccgcaag cccggtgcct gaacgcgtta agtcgacaat caacctctgg
    12901 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
    12961 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
    13021 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
    13081 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
    13141 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
    13201 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
    13261 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
    13321 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
    13381 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
    13441 agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta
    13501 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
    13561 tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca
    13621 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
    13681 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
    13741 atccctcaga cccttttagt cagtgtggaa aatctctagc agggcccgtt taaacccgct
    13801 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
    13861 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
    13921 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
    13981 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
    14041 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
    14101 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
    14161 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
    14221 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
    14281 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
    14341 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
    14401 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
    14461 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcg
//
