LOCUS       sequence-333282-       14507 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid allows efficient KO of the cldn1 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 CLDN1 2xgRNA - spCas9 iRFP670 puro
FEATURES             Location/Qualifiers
     source          1..14507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2621..2861
                     /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     2621..2641
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2792..2811
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2884..2959
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2973..3187
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     3108..3127
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        3208..3283
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3344..3555
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3563..3572
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3572..3637
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3644..3664
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3689..7789
                     /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             3689..7789
                     /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             7790..7837
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7844..7897
                     /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             7898..7948
                     /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      7964..7973
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7970..8902
                     /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             8930..8986
                     /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             8987..9583
                     /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     9480..9500
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9599..10187
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9652..9672)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10070..10081)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10259..10492
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10518..10535)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10524..10748
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10794..11222
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10881..10900)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11091..11112
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11231..11251)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11236..11565
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11416..11551
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11478..11497
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11613..11660
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11679..12053
                     /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    12183..12304
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12220..12239)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12274..12293
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12353..12369)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12353..12369)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12366..12388)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12377..12393
                     /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(12401..12407,12408..12425,12426..12431))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12446..12467
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12584..12601)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12755..13343)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12835..12854)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13514..14305,14306..14374))
                     /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     14137..14156
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14375..14479)
                     /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 agataaaggt cgggcaggaa gagggcctat ttcccatgat
     2641 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac
     2701 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     2761 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     2821 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgggagcgag tcatggccaa
     2881 cgcgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     2941 agtggcaccg agtcggtgct ttttttctcg aggaacgctg acgtcatcaa cccgctccaa
     3001 ggaatcgcgg gcccagtgtc actaggcggg aacacccagc gcgcgtgcgc cctggcagga
     3061 agatggctgt gagggacagg ggagtggcgc cctgcaatat ttgcatgtcg ctatgtgttc
     3121 tgggaaatca ccataaacgt gaaatgtctt tggatttggg aatcttataa gttctgtatg
     3181 agaccaccaa cagctgcagc cccgcgtgtt ttagagctag aaatagcaag ttaaaataag
     3241 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt acaatctagc
     3301 taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg
     3361 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc ctagagaagg
     3421 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
     3481 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
     3541 gccgccagaa cacaggaccg gtgccaccat ggactataag gaccacgacg gagactacaa
     3601 ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa
     3661 ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg
     3721 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     3781 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     3841 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     3901 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     3961 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     4021 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     4081 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     4141 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     4201 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     4261 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     4321 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     4381 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     4441 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     4501 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     4561 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     4621 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     4681 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     4741 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     4801 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     4861 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     4921 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     4981 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     5041 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     5101 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     5161 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     5221 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     5281 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     5341 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     5401 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     5461 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     5521 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     5581 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     5641 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     5701 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     5761 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     5821 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     5881 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     5941 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     6001 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     6061 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     6121 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     6181 gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat
     6241 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc
     6301 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     6361 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     6421 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     6481 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     6541 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     6601 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     6661 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     6721 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     6781 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     6841 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     6901 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     6961 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     7021 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     7081 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     7141 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     7201 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     7261 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     7321 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     7381 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     7441 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     7501 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     7561 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     7621 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     7681 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
     7741 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc
     7801 ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggaa ttcgagggca gaggaagtct
     7861 gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg ggcgtggccg acttgatcaa
     7921 gaagttcgag tccatctcca aggaggaggg ggatccaccg gtcgccacca tggcgcgtaa
     7981 ggtcgatctc acctcctgcg atcgcgagcc gatccacatc cccggcagca ttcagccgtg
     8041 cggctgcctg ctagcctgcg acgcgcaggc ggtgcggatc acgcgcatta cggaaaatgc
     8101 cggcgcgttc tttggacgcg aaactccgcg ggtcggtgag ctactcgccg attacttcgg
     8161 cgagaccgaa gcccatgcgc tgcgcaacgc actggcgcag tcctccgatc caaagcgacc
     8221 ggcgctgatc ttcggttggc gcgacggcct gaccggccgc accttcgaca tctcactgca
     8281 tcgccatgac ggtacatcga tcatcgagtt cgagcctgcg gcggccgaac aggccgacaa
     8341 tccgctgcgg ctgacgcggc agatcatcgc gcgcaccaaa gaactgaagt cgctcgaaga
     8401 gatggccgca cgggtgccgc gctatctgca ggcgatgctc ggctatcacc gcgtgatgtt
     8461 gtaccgcttc gcggacgacg gctccgggat ggtgatcggc gaggcgaagc gcagcgacct
     8521 cgagagcttt ctcggtcagc actttccggc gtcgctggtc ccgcagcagg cgcggctact
     8581 gtacttgaag aacgcgatcc gcgtggtctc ggattcgcgc ggcatcagca gccggatcgt
     8641 gcccgagcac gacgcctccg gcgccgcgct cgatctgtcg ttcgcgcacc tgcgcagcat
     8701 ctcgccctgc catctcgaat ttctgcggaa catgggcgtc agcgcctcga tgtcgctgtc
     8761 gatcatcatt gacggcacgc tatggggatt gatcatctgt catcattacg agccgcgtgc
     8821 cgtgccgatg gcgcagcgcg tcgcggccga aatgttcgcc gacttcttat cgctgcactt
     8881 caccgccgcc caccaccaac gctctagaag cgctggtacc ggatccggcg caacaaactt
     8941 ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgaccg agtacaagcc
     9001 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     9061 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     9121 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     9181 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     9241 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     9301 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     9361 caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     9421 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     9481 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     9541 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa
     9601 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     9661 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     9721 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     9781 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     9841 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     9901 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     9961 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
    10021 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
    10081 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
    10141 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga
    10201 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
    10261 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
    10321 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
    10381 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
    10441 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagggcccgt
    10501 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
    10561 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
    10621 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
    10681 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
    10741 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
    10801 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
    10861 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
    10921 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
    10981 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
    11041 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
    11101 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
    11161 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
    11221 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
    11281 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
    11341 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
    11401 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
    11461 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
    11521 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
    11581 tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata
    11641 tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg
    11701 ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg
    11761 ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc
    11821 tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg
    11881 tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg
    11941 acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc
    12001 tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc
    12061 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
    12121 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
    12181 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
    12241 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
    12301 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
    12361 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
    12421 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
    12481 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
    12541 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
    12601 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
    12661 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
    12721 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
    12781 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
    12841 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
    12901 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
    12961 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
    13021 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
    13081 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
    13141 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
    13201 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
    13261 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
    13321 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
    13381 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
    13441 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
    13501 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
    13561 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
    13621 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
    13681 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
    13741 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
    13801 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
    13861 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
    13921 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
    13981 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
    14041 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
    14101 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
    14161 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
    14221 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
    14281 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
    14341 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
    14401 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
    14461 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgac
//
