LOCUS       sequence-417652-       14507 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  codes for 2 gRNA-TracrRNA targeting human OCLN, Cas9, iRFP670 
            fluorescent protein and puromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14507)
  AUTHORS   Baptiste Pradel , Maika S. Deffieu, Veronique Robert-Hebmann, 
            Guilhem Cantaloube, Mathias Faure, Nathalie Chazal, View ORCID 
            ProfileRaphael Gaudin, View ORCID ProfileLucile Espert
  TITLE     LC3B conjugation machinery promotes autophagy-independent HIV-1 
            entry in CD4+ T lymphocytes
  JOURNAL   bioRxiv
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: "bioRxiv"
COMMENT     Sequence Label: pLenti gRNA KO OCLNx2 spCas9-T2A-iRFP670-P2A-puro
FEATURES             Location/Qualifiers
     source          1..14507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(94..169)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(184..432)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(242..261)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(412..432)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    496..613
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(911..955)
                     /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    1140..1373
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    1866..1991
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             2038..2218
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(2236..2434)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2261..2281)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2436..2815
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2987..3006
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3081..3185
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3186..3254,3255..4046)
                     /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     complement(3404..3423)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4217..4805
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4706..4725
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4959..4976
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5093..5114
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5129..5134,5135..5152,5153..5159)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5167..5183
                     /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)."
     primer_bind     5172..5194
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5191..5207
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5191..5207
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5256..5377
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5267..5286)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5321..5340
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5507..5881)
                     /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"
     promoter        complement(5900..5947)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5995..6324)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6009..6144
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6063..6082)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     6309..6329
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(6338..6766)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6448..6469)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6660..6679
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    6812..7036
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     7025..7042
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     LTR             7068..7301
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    7373..7961
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             7479..7490
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7888..7908
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7977..8573)
                     /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     complement(8060..8080)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             complement(8574..8630)
                     /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             complement(8658..9590)
                     /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"
     regulatory      9587..9596
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(9612..9662)
                     /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"
     CDS             complement(9663..9716)
                     /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             complement(9723..9770)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(9771..13871)
                     /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             complement(9771..13871)
                     /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             complement(13896..13916)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(13923..13988)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      13988..13997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(14005..14216)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_RNA        complement(14277..14352)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(14373..14507,1..80))
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(14433..14452)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
ORIGIN
        1 gggcgcacgc gcgctgggtg ttcccgccta gtgacactgg gcccgcgatt ccttggagcg
       61 ggttgatgac gtcagcgttc ctcgagaaaa aaagcaccga ctcggtgcca ctttttcaag
      121 ttgataacgg actagcctta ttttaacttg ctatttctag ctctaaaaca ggtggacttt
      181 caagaggccc cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt
      241 tacggtaagc atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag
      301 aaattattac tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt
      361 aattccaatt atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg
      421 aaataggccc tcttcctgcc cgacctttat cttaattaac caaactggat ctctgctgtc
      481 cctgtaataa acccgaaaat tttgaatttt tgtaatttgt ttttgtaatt ctttagtttg
      541 tatgtctgtt gctattatgt ctactattct ttcccctgca ctgtaccccc caatcccccc
      601 ttttctttta aaattgtgga tgaatactgc catttgtctg cagaattggc gcacgcagtg
      661 ccgatccgtt cactaatcga atggatctgt ctctgtctct ctctccacct tcttcttcta
      721 ttccttcggg cctgtcgggt cccctcgggg ttgggaggtg ggtctgaaac gataatggtg
      781 aatatccctg cctaactcta ttcactatag aaagtacagc aaaaactatt cttaaaccta
      841 ccaagcctcc tactatcatt atgaataatt ttatatacca cagccaattt gttatgttaa
      901 accaattcca caaacttgcc catttatcta attccaataa ttcttgttca ttcttttctt
      961 gctggttttg cgattcttca attaaggagt gtattaagct tgtgtaattg ttaatttctc
     1021 tgtcccactc catccaggtc gtgtgattcc aaatctgttc cagagattta ttactccaac
     1081 tagcattcca aggcacagca gtggtgcaaa tgagttttcc agagcaaccc caaatcccca
     1141 ggagctgttg atcctttagg tatctttcca cagccaggat tcttgcctgg agctgcttga
     1201 tgccccagac tgtgagttgc aacagatgct gttgcgcctc aatagccctc agcaaattgt
     1261 tctgctgctg cactatacca gacaataatt gtctggcctg taccgtcagc gtcattgacg
     1321 ctgcgcccat agtgcttcct gctgctccca agaacccaag gaacaaagct cctattccca
     1381 ctgctctttt ttctctctgc accactcttc tctttgcctt ggtgggtgct actcctaatg
     1441 gttcaatttt tactacttta tatttatata attcacttct ccaattgtcc ctcatatctc
     1501 ctcctccagg tctgaagatc agcggccgct tgctgtgcgg tggtcttact tttgttttgc
     1561 tcttcctcta tcttgtctaa agcttccttg gtgtctttta tctctatcct ttgatgcaca
     1621 caatagaggg ttgctactgt attatataat gatctaagtt cttctgatcc tgtctgaagg
     1681 gatggttgta gctgtcccag tatttgtcta cagccttctg atgtttctaa caggccagga
     1741 ttaactgcga atcgttctag ctccctgctt gcccatacta tatgttttaa tttatatttt
     1801 ttctttcccc ctggccttaa ccgaattttt tcccatcgcg atctaattct cccccgctta
     1861 atactgacgc tctcgcaccc atctctctcc ttctagcctc cgctagtcaa aatttttggc
     1921 gtactcacca gtcgccgccc ctcgcctctt gccgtgcgcg cttcagcaag ccgagtcctg
     1981 cgtcgagaga gctcctctgg tttccctttc gctttcaagt ccctgttcgg gcgccactgc
     2041 tagagatttt ccacactgac taaaagggtc tgagggatct ctagttacca gagtcacaca
     2101 acagacgggc acacactact tgaagcactc aaggcaagct ttattgaggc ttaagcagtg
     2161 ggttccctag ttagccagag agctcccagg ctcagatctg gtctaaccag agagacccag
     2221 tacaggcaaa acgcgctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc
     2281 gtcaatgggg cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac
     2341 aaactcccat tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat
     2401 ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg
     2461 tagatgtact gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg
     2521 ggccatttac cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt
     2581 actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct
     2641 attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc
     2701 ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta
     2761 tgaactaatg accccgtaat tgattactat taataactag tcaataatca atgtcaacgc
     2821 gtatatctgg cccgtacatc gcgaagcagc gcaaaacgcc taaccctaag cagattcttc
     2881 atgcaattgt cggtcaagcc ttgccttgtt gtagcttaaa ttttgctcgc gcactactca
     2941 gcgacctcca acacacaagc agggagcaga tactggctta actatgcggc atcagagcag
     3001 attgtactga gagtgcacca taggggatcg ggagatctcc cgatccgtcg acgtcaggtg
     3061 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3121 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3181 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     3241 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     3301 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     3361 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     3421 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     3481 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     3541 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     3601 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     3661 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     3721 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3781 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3841 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     3901 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     3961 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4021 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     4081 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     4141 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4201 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4261 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4321 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     4381 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4441 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4501 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4561 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4621 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4681 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4741 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4801 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4861 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4921 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4981 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     5041 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     5101 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     5161 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     5221 agctctagct agaggtcgac ggtatacaga catgataaga tacattgatg agtttggaca
     5281 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     5341 tttatttgta accattataa gctgcaataa acaagttggg gtgggcgaag aactccagca
     5401 tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca
     5461 acctttcata gaaggcggcg gtggaatcga aatctcgtag cacgtgtcag tcctgctcct
     5521 cggccacgaa gtgcacgcag ttgccggccg ggtcgcgcag ggcgaactcc cgcccccacg
     5581 gctgctcgcc gatctcggtc atggccggcc cggaggcgtc ccggaagttc gtggacacga
     5641 cctccgacca ctcggcgtac agctcgtcca ggccgcgcac ccacacccag gccagggtgt
     5701 tgtccggcac cacctggtcc tggaccgcgc tgatgaacag ggtcacgtcg tcccggacca
     5761 caccggcgaa gtcgtcctcc acgaagtccc gggagaaccc gagccggtcg gtccagaact
     5821 cgaccgctcc ggcgacgtcg cgcgcggtga gcaccggaac ggcactggtc aacttggcca
     5881 tggtttagtt cctcaccttg tcgtattata ctatgccgat atactatgcc gatgattaat
     5941 tgtcaacacg tgctgatcag atccgaaaat ggatatacaa gctcccggga gctttttgca
     6001 aaagcctagg cctccaaaaa agcctcctca ctacttctgg aatagctcag aggcagaggc
     6061 ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca tggggcggag aatgggcgga
     6121 actgggcgga gttaggggcg ggatgggcgg agttaggggc gggactatgg ttgctgacta
     6181 attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact ttccacacct
     6241 ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga
     6301 ctttccacac cctaactgac acacattcca cagaattaat tcgcgttaaa tttttgttaa
     6361 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     6421 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     6481 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     6541 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     6601 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     6661 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     6721 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtgggg atacccccta
     6781 gagccccagc tggttctttc cgcctcagaa gccatagagc ccaccgcatc cccagcatgc
     6841 ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc ccccagaata
     6901 gaatgacacc tactcagaca atgcgatgca atttcctcat tttattagga aaggacagtg
     6961 ggagtggcac cttccagggt caaggaaggc acgggggagg ggcaaacaac agatggctgg
     7021 caactagaag gcacagtcga ggctgatcag cgggtttaaa cgggccctgc tagagatttt
     7081 ccacactgac taaaagggtc tgagggatct ctagttacca gagtcacaca acagacgggc
     7141 acacactact tgaagcactc aaggcaagct ttattgaggc ttaagcagtg ggttccctag
     7201 ttagccagag agctcccagg ctcagatctg gtctaaccag agagacccag tacaagcaaa
     7261 aagcagatct tgtcttcgtt gggagtgaat tagcccttcc agtcccccct tttcttttaa
     7321 aaagtggcta agatctacag ctgccttgta agtcattggt cttaaagtcg acgcggggag
     7381 gcggcccaaa gggagatccg actcgtctga gggcgaaggc gaagacgcgg aagaggccgc
     7441 agagccggca gcaggccgcg ggaaggaagg tccgctggat tgagggccga agggacgtag
     7501 cagaaggacg tcccgcgcag aatccaggtg gcaacacagg cgagcagcca aggaaaggac
     7561 gatgatttcc ccgacaacac cacggaattg tcagtgccca acagccgagc ccctgtccag
     7621 cagcgggcaa ggcaggcggc gatgagttcc gccgtggcaa tagggagggg gaaagcgaaa
     7681 gtcccggaaa ggagctgaca ggtggtggca atgccccaac cagtgggggt tgcgtcagca
     7741 aacacagtgc acaccacgcc acgttgcctg acaacgggcc acaactcctc ataaagagac
     7801 agcaaccagg atttatacaa ggaggagaaa atgaaagcca tacgggaagc aatagcatga
     7861 tacaaaggca ttaaagcagc gtatccacat agcgtaaaag gagcaacata gttaagaata
     7921 ccagtcaatc tttcacaaat tttgtaatcc agaggttgat tgtcgactta acgcgttcag
     7981 gcaccgggct tgcgggtcat gcaccaggtg cgcggtcctt cgggcacctc gacgtcggcg
     8041 gtgacggtga agccgagccg ctcgtagaag gggaggttgc ggggcgcgga ggtctccagg
     8101 aaggcgggca ccccggcgcg ctcggccgcc tccactccgg ggagcacgac ggcgctgccc
     8161 agacccttgc cctggtggtc gggcgagact ccgacggtgg ccaggaacca cgcgggctcc
     8221 ttgggccggt gcggcgccag gaggccttcc atctgttgct gcgcggccag ccgggaaccg
     8281 ctcaactcgg ccatgcgcgg gccgatctcg gcgaacaccg cccccgcttc gacgctctcc
     8341 ggcgtggtcc agaccgccac cgcggcgccg tcgtccgcga cccacacctt gccgatgtcg
     8401 agcccgacgc gcgtgaggaa gagttcttgc agctcggtga cccgctcgat gtggcggtcc
     8461 ggatcgacgg tgtggcgcgt ggcggggtag tcggcgaacg cggcggcgag ggtgcgtacg
     8521 gccctgggga cgtcgtcgcg ggtggcgagg cgcaccgtgg gcttgtactc ggtcggtcca
     8581 ggattctctt cgacatctcc ggcttgtttc agcagagaga agtttgttgc gccggatccg
     8641 gtaccagcgc ttctagagcg ttggtggtgg gcggcggtga agtgcagcga taagaagtcg
     8701 gcgaacattt cggccgcgac gcgctgcgcc atcggcacgg cacgcggctc gtaatgatga
     8761 cagatgatca atccccatag cgtgccgtca atgatgatcg acagcgacat cgaggcgctg
     8821 acgcccatgt tccgcagaaa ttcgagatgg cagggcgaga tgctgcgcag gtgcgcgaac
     8881 gacagatcga gcgcggcgcc ggaggcgtcg tgctcgggca cgatccggct gctgatgccg
     8941 cgcgaatccg agaccacgcg gatcgcgttc ttcaagtaca gtagccgcgc ctgctgcggg
     9001 accagcgacg ccggaaagtg ctgaccgaga aagctctcga ggtcgctgcg cttcgcctcg
     9061 ccgatcacca tcccggagcc gtcgtccgcg aagcggtaca acatcacgcg gtgatagccg
     9121 agcatcgcct gcagatagcg cggcacccgt gcggccatct cttcgagcga cttcagttct
     9181 ttggtgcgcg cgatgatctg ccgcgtcagc cgcagcggat tgtcggcctg ttcggccgcc
     9241 gcaggctcga actcgatgat cgatgtaccg tcatggcgat gcagtgagat gtcgaaggtg
     9301 cggccggtca ggccgtcgcg ccaaccgaag atcagcgccg gtcgctttgg atcggaggac
     9361 tgcgccagtg cgttgcgcag cgcatgggct tcggtctcgc cgaagtaatc ggcgagtagc
     9421 tcaccgaccc gcggagtttc gcgtccaaag aacgcgccgg cattttccgt aatgcgcgtg
     9481 atccgcaccg cctgcgcgtc gcaggctagc aggcagccgc acggctgaat gctgccgggg
     9541 atgtggatcg gctcgcgatc gcaggaggtg agatcgacct tacgcgccat ggtggcgacc
     9601 ggtggatccc cctcctcctt ggagatggac tcgaacttct tgatcaagtc ggccacgccc
     9661 attgggccag gattctcctc gacgtcaccg catgttagca gacttcctct gccctcgaat
     9721 tcctttttct tttttgcctg gccggccttt ttcgtggccg ccggcctttt gtcgcctccc
     9781 agctgagaca ggtcgatccg tgtctcgtac aggccggtga tgctctggtg gatcagggtg
     9841 gcgtccagca cctctttggt gctggtgtac ctcttccggt cgatggtggt gtcaaagtac
     9901 ttgaaggcgg caggggctcc cagattggtc agggtaaaca ggtggatgat attctcggcc
     9961 tgctctctga tgggcttatc ccggtgcttg ttgtaggcgg acagcacttt gtccagatta
    10021 gcgtcggcca ggatcactct cttggagaac tcgctgatct gctcgatgat ctcgtccagg
    10081 tagtgcttgt gctgttccac aaacagctgt ttctgctcat tatcctcggg ggagcccttc
    10141 agcttctcat agtggctggc caggtacagg aagttcacat atttggaggg cagggccagt
    10201 tcgtttccct tctgcagttc gccggcagag gccagcattc tcttccggcc gttttccagc
    10261 tcgaacaggg agtacttagg cagcttgatg atcaggtcct ttttcacttc tttgtagccc
    10321 ttggcttcca gaaagtcgat gggattcttc tcgaagctgc ttctttccat gatggtgatc
    10381 cccagcagct ctttcacact cttcagtttc ttggacttgc ccttttccac tttggccacc
    10441 accagcacag aataggccac ggtggggctg tcgaagccgc cgtacttctt agggtcccag
    10501 tccttctttc tggcgatcag cttatcgctg ttcctcttgg gcaggataga ctctttgctg
    10561 aagccgcctg tctgcacctc ggtctttttc acgatattca cttggggcat gctcagcact
    10621 ttccgcacgg tggcaaaatc ccggccctta tcccacacga tctccccggt ttcgccgttt
    10681 gtctcgatca gaggccgctt ccggatctcg ccgttggcca gggtaatctc ggtcttgaaa
    10741 aagttcatga tgttgctgta gaagaagtac ttggcggtag ccttgccgat ttcctgctcg
    10801 ctcttggcga tcatcttccg cacgtcgtac accttgtagt cgccgtacac gaactcgctt
    10861 tccagcttag ggtacttttt gatcagggcg gttcccacga cggcgttcag gtaggcgtcg
    10921 tgggcgtggt ggtagttgtt gatctcgcgc actttgtaaa actggaaatc cttccggaaa
    10981 tcggacacca gcttggactt cagggtgatc actttcactt cccggatcag cttgtcattc
    11041 tcgtcgtact tagtgttcat ccgggagtcc aggatctgtg ccacgtgctt tgtgatctgc
    11101 cgggtttcca ccagctgtct cttgatgaag ccggccttat ccagttcgct caggccgcct
    11161 ctctcggcct tggtcagatt gtcgaacttt ctctgggtaa tcagcttggc gttcagcagc
    11221 tgccgccagt agttcttcat cttcttcacg acctcttcgg agggcacgtt gtcgctcttg
    11281 ccccggttct tgtcgcttct ggtcagcacc ttgttgtcga tggagtcgtc cttcagaaag
    11341 ctctgaggca cgatatggtc cacatcgtag tcggacagcc ggttgatgtc cagttcctgg
    11401 tccacgtaca tatcccgccc attctgcagg tagtacaggt acagcttctc gttctgcagc
    11461 tgggtgtttt ccacggggtg ttctttcagg atctggctgc ccagctcttt gatgccctct
    11521 tcgatccgct tcattctctc gcggctgttc ttctgtccct tctgggtggt ctggttctct
    11581 ctggccattt cgatcacgat gttctcgggc ttgtgccggc ccatcacttt cacgagctcg
    11641 tccaccacct tcactgtctg caggatgccc ttcttaatgg cggggctgcc ggccagattg
    11701 gcaatgtgct cgtgcaggct atcgccctgg ccggacacct gggctttctg gatgtcctct
    11761 ttaaaggtca ggctgtcgtc gtggatcagc tgcatgaagt ttctgttggc gaagccgtcg
    11821 gacttcagga aatccaggat tgtcttgccg gactgcttgt cccggatgcc gttgatcagc
    11881 ttccggctca gcctgcccca gccggtgtat ctccgccgct tcagctgctt catcactttg
    11941 tcgtcgaaca ggtgggcata ggttttcagc cgttcctcga tcatctctct gtcctcaaac
    12001 agtgtcaggg tcagcacgat atcttccaga atgtcctcgt tttcctcatt gtccaggaag
    12061 tccttgtcct tgataatttt cagcagatcg tggtatgtgc ccagggaggc gttgaaccga
    12121 tcttccacgc cggagatttc cacggagtcg aagcactcga ttttcttgaa gtagtcctct
    12181 ttcagctgct tcacggtcac tttccggttg gtcttgaaca gcaggtccac gatggccttt
    12241 ttctgctcgc cgctcaggaa ggcgggcttt ctcattccct cggtcacgta tttcactttg
    12301 gtcagctcgt tatacacggt gaagtactcg tacagcaggc tgtgcttggg cagcaccttc
    12361 tcgttgggca ggttcttatc gaagttggtc atccgctcga tgaagctctg ggcggaagcg
    12421 cccttgtcca ccacttcctc gaagttccag ggggtgatgg tttcctcgct ctttctggtc
    12481 atccaggcga atctgctgtt tcccctggcc agagggccca cgtagtaggg gatgcggaag
    12541 gtcaggatct tctcgatctt ttcccggttg tccttcagga atgggtaaaa atcttcctgc
    12601 cgccgcagaa tggcgtgcag ctctcccagg tggatctggt gggggatgct gccgttgtcg
    12661 aaggtccgct gcttccgcag caggtcctct ctgttcagct tcacgagcag ttcctcggtg
    12721 ccgtccatct tttccaggat gggcttgatg aacttgtaga actcttcctg gctggctccg
    12781 ccgtcaatgt agccggcgta gccgttcttg ctctggtcga agaaaatctc tttgtacttc
    12841 tcaggcagct gctgccgcac gagagctttc agcagggtca ggtcctggtg gtgctcgtcg
    12901 tatctcttga tcatagaggc gctcaggggg gccttggtga tctcggtgtt cactctcagg
    12961 atgtcgctca gcaggatggc gtcggacagg ttcttggcgg ccagaaacag gtcggcgtac
    13021 tggtcgccga tctgggccag caggttgtcc aggtcgtcgt cgtaggtgtc cttgctcagc
    13081 tgcagtttgg catcctcggc caggtcgaag ttgctcttga agttgggggt caggcccagg
    13141 ctcagggcaa tcaggtttcc gaacaggcca ttcttcttct cgccgggcag ctgggcgatc
    13201 agattttcca gccgtctgct cttgctcagt ctggcagaca ggatggcctt ggcgtccacg
    13261 ccgctggcgt tgatggggtt ttcctcgaac agctggttgt aggtctgcac cagctggatg
    13321 aacagcttgt ccacgtcgct gttgtcgggg ttcaggtcgc cctcgatcag gaagtggccc
    13381 cggaacttga tcatgtgggc cagggccaga tagatcagcc gcaggtcggc cttgtcggtg
    13441 ctgtccacca gtttctttct caggtggtag atggtggggt acttctcgtg gtaggccacc
    13501 tcgtccacga tgttgccgaa gatggggtgc cgctcgtgct tcttatcctc ttccaccagg
    13561 aaggactctt ccagtctgtg gaagaagctg tcgtccacct tggccatctc gttgctgaag
    13621 atctcttgca gatagcagat ccggttcttc cgtctggtgt atcttcttct ggcggttctc
    13681 ttcagccggg tggcctcggc tgtttcgccg ctgtcgaaca gcagggctcc gatcaggttc
    13741 ttcttgatgc tgtgccggtc ggtgttgccc agcaccttga atttcttgct gggcaccttg
    13801 tactcgtcgg tgatcacggc ccagcccaca gagttggtgc cgatgtccag gccgatgctg
    13861 tacttcttgt cggctgctgg gactccgtgg ataccgacct tccgcttctt ctttggggcc
    13921 atcttatcgt catcgtcttt gtaatcaata tcatgatcct tgtagtctcc gtcgtggtcc
    13981 ttatagtcca tggtggcacc ggtcctgtgt tctggcggca aacccgttgc gaaaaagaac
    14041 gttcacggcg actactgcac ttatatacgg ttctccccca ccctcgggaa aaaggcggag
    14101 ccagtacacg acatcacttt cccagtttac cccgcgccac cttctctagg caccggatca
    14161 attgccgacc cctcccccca acttctcggg gactgtgggc gatgtgcgct ctgcccactg
    14221 acgggcaccg gagccaattc ccactccttt caagacctag ctagattgta aaaaaagcac
    14281 cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac ttgctatttc
    14341 tagctctaaa acttcaagag gcctggatga cagtggtctc atacagaact tataagattc
    14401 ccaaatccaa agacatttca cgtttatggt gatttcccag aacacatagc gacatgcaaa
    14461 tattgcaggg cgccactccc ctgtccctca cagccatctt cctgcca
//
