LOCUS       sequence-417651-       14154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  codes for 2 gRNA-TracrRNA targeting human OCLN, Cas9, E2-Crimson 
            fluorescent protein and puromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14154)
  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 14154)
  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-Crimson-P2A-puro
FEATURES             Location/Qualifiers
     source          1..14154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(15..255)
                     /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     complement(65..84)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(235..255)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    319..436
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(734..778)
                     /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    963..1196
                     /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    1689..1814
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             1861..2041
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(2059..2257)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2084..2104)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2259..2638
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2810..2829
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2904..3008
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3009..3077,3078..3869)
                     /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(3227..3246)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4040..4628
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4529..4548
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4782..4799
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4916..4937
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4952..4957,4958..4975,4976..4982)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4990..5006
                     /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     4995..5017
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5014..5030
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5014..5030
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5079..5200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5090..5109)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5144..5163
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5330..5704)
                     /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(5723..5770)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5818..6147)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5832..5967
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5886..5905)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     6132..6152
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(6161..6589)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6271..6292)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6483..6502
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    6635..6859
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6848..6865
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     LTR             6891..7124
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    7196..7784
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             7302..7313
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7711..7731
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7800..8396)
                     /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(7883..7903)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             complement(8397..8453)
                     /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(8481..9152)
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2009)"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="DSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAKL
                     QVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHMA
                     LKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHHL
                     FQ"
     primer_bind     complement(8738..8757)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             complement(9153..9206)
                     /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(9213..9260)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(9261..13361)
                     /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(9261..13361)
                     /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(13386..13406)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(13413..13478)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      13478..13487
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(13495..13706)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        complement(13853..14067)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(13913..13932)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
ORIGIN
        1 tttcaagagg ccccgtcctt tccacaagat atataaagcc aagaaatcga aatactttca
       61 agttacggta agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc
      121 aagaaattat tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa
      181 attaattcca attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat
      241 gggaaatagg ccctcttcct gcccgacctt tatcttaatt aaccaaactg gatctctgct
      301 gtccctgtaa taaacccgaa aattttgaat ttttgtaatt tgtttttgta attctttagt
      361 ttgtatgtct gttgctatta tgtctactat tctttcccct gcactgtacc ccccaatccc
      421 cccttttctt ttaaaattgt ggatgaatac tgccatttgt ctgcagaatt ggcgcacgca
      481 gtgccgatcc gttcactaat cgaatggatc tgtctctgtc tctctctcca ccttcttctt
      541 ctattccttc gggcctgtcg ggtcccctcg gggttgggag gtgggtctga aacgataatg
      601 gtgaatatcc ctgcctaact ctattcacta tagaaagtac agcaaaaact attcttaaac
      661 ctaccaagcc tcctactatc attatgaata attttatata ccacagccaa tttgttatgt
      721 taaaccaatt ccacaaactt gcccatttat ctaattccaa taattcttgt tcattctttt
      781 cttgctggtt ttgcgattct tcaattaagg agtgtattaa gcttgtgtaa ttgttaattt
      841 ctctgtccca ctccatccag gtcgtgtgat tccaaatctg ttccagagat ttattactcc
      901 aactagcatt ccaaggcaca gcagtggtgc aaatgagttt tccagagcaa ccccaaatcc
      961 ccaggagctg ttgatccttt aggtatcttt ccacagccag gattcttgcc tggagctgct
     1021 tgatgcccca gactgtgagt tgcaacagat gctgttgcgc ctcaatagcc ctcagcaaat
     1081 tgttctgctg ctgcactata ccagacaata attgtctggc ctgtaccgtc agcgtcattg
     1141 acgctgcgcc catagtgctt cctgctgctc ccaagaaccc aaggaacaaa gctcctattc
     1201 ccactgctct tttttctctc tgcaccactc ttctctttgc cttggtgggt gctactccta
     1261 atggttcaat ttttactact ttatatttat ataattcact tctccaattg tccctcatat
     1321 ctcctcctcc aggtctgaag atcagcggcc gcttgctgtg cggtggtctt acttttgttt
     1381 tgctcttcct ctatcttgtc taaagcttcc ttggtgtctt ttatctctat cctttgatgc
     1441 acacaataga gggttgctac tgtattatat aatgatctaa gttcttctga tcctgtctga
     1501 agggatggtt gtagctgtcc cagtatttgt ctacagcctt ctgatgtttc taacaggcca
     1561 ggattaactg cgaatcgttc tagctccctg cttgcccata ctatatgttt taatttatat
     1621 tttttctttc cccctggcct taaccgaatt ttttcccatc gcgatctaat tctcccccgc
     1681 ttaatactga cgctctcgca cccatctctc tccttctagc ctccgctagt caaaattttt
     1741 ggcgtactca ccagtcgccg cccctcgcct cttgccgtgc gcgcttcagc aagccgagtc
     1801 ctgcgtcgag agagctcctc tggtttccct ttcgctttca agtccctgtt cgggcgccac
     1861 tgctagagat tttccacact gactaaaagg gtctgaggga tctctagtta ccagagtcac
     1921 acaacagacg ggcacacact acttgaagca ctcaaggcaa gctttattga ggcttaagca
     1981 gtgggttccc tagttagcca gagagctccc aggctcagat ctggtctaac cagagagacc
     2041 cagtacaggc aaaacgcgct gcttatatag acctcccacc gtacacgcct accgcccatt
     2101 tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa
     2161 aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc
     2221 tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat
     2281 acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag
     2341 gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga
     2401 tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc
     2461 cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg
     2521 ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg
     2581 ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa tcaatgtcaa
     2641 cgcgtatatc tggcccgtac atcgcgaagc agcgcaaaac gcctaaccct aagcagattc
     2701 ttcatgcaat tgtcggtcaa gccttgcctt gttgtagctt aaattttgct cgcgcactac
     2761 tcagcgacct ccaacacaca agcagggagc agatactggc ttaactatgc ggcatcagag
     2821 cagattgtac tgagagtgca ccatagggga tcgggagatc tcccgatccg tcgacgtcag
     2881 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2941 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3001 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3061 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3121 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3181 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3241 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3301 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3361 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3421 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3481 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3541 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3601 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3661 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3721 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3781 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3841 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3901 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3961 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4021 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4081 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4141 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     4201 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4261 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4321 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4381 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4441 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4501 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4561 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4621 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4681 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4741 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4801 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     4861 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     4921 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     4981 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     5041 ccaagctcta gctagaggtc gacggtatac agacatgata agatacattg atgagtttgg
     5101 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     5161 tgctttattt gtaaccatta taagctgcaa taaacaagtt ggggtgggcg aagaactcca
     5221 gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg attccgaagc
     5281 ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tagcacgtgt cagtcctgct
     5341 cctcggccac gaagtgcacg cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc
     5401 acggctgctc gccgatctcg gtcatggccg gcccggaggc gtcccggaag ttcgtggaca
     5461 cgacctccga ccactcggcg tacagctcgt ccaggccgcg cacccacacc caggccaggg
     5521 tgttgtccgg caccacctgg tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga
     5581 ccacaccggc gaagtcgtcc tccacgaagt cccgggagaa cccgagccgg tcggtccaga
     5641 actcgaccgc tccggcgacg tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg
     5701 ccatggttta gttcctcacc ttgtcgtatt atactatgcc gatatactat gccgatgatt
     5761 aattgtcaac acgtgctgat cagatccgaa aatggatata caagctcccg ggagcttttt
     5821 gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggcaga
     5881 ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc
     5941 ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
     6001 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     6061 cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg
     6121 ggactttcca caccctaact gacacacatt ccacagaatt aattcgcgtt aaatttttgt
     6181 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     6241 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     6301 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     6361 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     6421 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     6481 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     6541 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtg gggatacccc
     6601 ctagagcccc agctggttct ttccgcctca gaagccatag agcccaccgc atccccagca
     6661 tgcctgctat tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga
     6721 atagaatgac acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca
     6781 gtgggagtgg caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc
     6841 tggcaactag aaggcacagt cgaggctgat cagcgggttt aaacgggccc tgctagagat
     6901 tttccacact gactaaaagg gtctgaggga tctctagtta ccagagtcac acaacagacg
     6961 ggcacacact acttgaagca ctcaaggcaa gctttattga ggcttaagca gtgggttccc
     7021 tagttagcca gagagctccc aggctcagat ctggtctaac cagagagacc cagtacaagc
     7081 aaaaagcaga tcttgtcttc gttgggagtg aattagccct tccagtcccc ccttttcttt
     7141 taaaaagtgg ctaagatcta cagctgcctt gtaagtcatt ggtcttaaag tcgacgcggg
     7201 gaggcggccc aaagggagat ccgactcgtc tgagggcgaa ggcgaagacg cggaagaggc
     7261 cgcagagccg gcagcaggcc gcgggaagga aggtccgctg gattgagggc cgaagggacg
     7321 tagcagaagg acgtcccgcg cagaatccag gtggcaacac aggcgagcag ccaaggaaag
     7381 gacgatgatt tccccgacaa caccacggaa ttgtcagtgc ccaacagccg agcccctgtc
     7441 cagcagcggg caaggcaggc ggcgatgagt tccgccgtgg caatagggag ggggaaagcg
     7501 aaagtcccgg aaaggagctg acaggtggtg gcaatgcccc aaccagtggg ggttgcgtca
     7561 gcaaacacag tgcacaccac gccacgttgc ctgacaacgg gccacaactc ctcataaaga
     7621 gacagcaacc aggatttata caaggaggag aaaatgaaag ccatacggga agcaatagca
     7681 tgatacaaag gcattaaagc agcgtatcca catagcgtaa aaggagcaac atagttaaga
     7741 ataccagtca atctttcaca aattttgtaa tccagaggtt gattgtcgac ttaacgcgtt
     7801 caggcaccgg gcttgcgggt catgcaccag gtgcgcggtc cttcgggcac ctcgacgtcg
     7861 gcggtgacgg tgaagccgag ccgctcgtag aaggggaggt tgcggggcgc ggaggtctcc
     7921 aggaaggcgg gcaccccggc gcgctcggcc gcctccactc cggggagcac gacggcgctg
     7981 cccagaccct tgccctggtg gtcgggcgag actccgacgg tggccaggaa ccacgcgggc
     8041 tccttgggcc ggtgcggcgc caggaggcct tccatctgtt gctgcgcggc cagccgggaa
     8101 ccgctcaact cggccatgcg cgggccgatc tcggcgaaca ccgcccccgc ttcgacgctc
     8161 tccggcgtgg tccagaccgc caccgcggcg ccgtcgtccg cgacccacac cttgccgatg
     8221 tcgagcccga cgcgcgtgag gaagagttct tgcagctcgg tgacccgctc gatgtggcgg
     8281 tccggatcga cggtgtggcg cgtggcgggg tagtcggcga acgcggcggc gagggtgcgt
     8341 acggccctgg ggacgtcgtc gcgggtggcg aggcgcaccg tgggcttgta ctcggtcggt
     8401 ccaggattct cttcgacatc tccggcttgt ttcagcagag agaagtttgt tgcgccggat
     8461 ccggtaccag cgcttctaga ctggaacagg tggtggcggg cctcggcgcg ctcgtactgc
     8521 tccaccacgg tgtagtcctc gttgtgggag gtgatgtcga gcttgtagtc cacgtagtgg
     8581 tagccgggca gcttcacggg cttcttggcc atgtagatgg acttgaactc acacaggtag
     8641 tggccgccgc ccttcagctt cagcgccatg tggttctcgc ccttcagcac gccgtcgcgg
     8701 gggtagttgc gctcagtgga gggctcccag cccagagtct tcttctgcat tacggggccg
     8761 tcggagggga agttcacgcc gatgaacttc acgtggtaga tgagggtgcc gtcctgcagg
     8821 gaggagtcct gggtcacggt caccacgccg ccgtcctcga agttcatcac gcgctcccac
     8881 ttgaagccct cggggaagga ctgcttgagg tagtcgggga tgtcggcggg gtgcttgatg
     8941 tacgccttgg agccgtagaa gaactggggg gacaggatgt cccaggcgaa gggcaggggg
     9001 ccgcccttgg tcacttgcag cttggcggtc tgggtgccct cgtagggctt gccctcgccc
     9061 acgccctcga tctcgaactc gtggccgttc acggagccct ccatgtgcac cttgaagcgc
     9121 atgaagggct tgatgacgtt ctcagtgcta tctgggccag gattctcctc gacgtcaccg
     9181 catgttagca gacttcctct gccctcgaat tcctttttct tttttgcctg gccggccttt
     9241 ttcgtggccg ccggcctttt gtcgcctccc agctgagaca ggtcgatccg tgtctcgtac
     9301 aggccggtga tgctctggtg gatcagggtg gcgtccagca cctctttggt gctggtgtac
     9361 ctcttccggt cgatggtggt gtcaaagtac ttgaaggcgg caggggctcc cagattggtc
     9421 agggtaaaca ggtggatgat attctcggcc tgctctctga tgggcttatc ccggtgcttg
     9481 ttgtaggcgg acagcacttt gtccagatta gcgtcggcca ggatcactct cttggagaac
     9541 tcgctgatct gctcgatgat ctcgtccagg tagtgcttgt gctgttccac aaacagctgt
     9601 ttctgctcat tatcctcggg ggagcccttc agcttctcat agtggctggc caggtacagg
     9661 aagttcacat atttggaggg cagggccagt tcgtttccct tctgcagttc gccggcagag
     9721 gccagcattc tcttccggcc gttttccagc tcgaacaggg agtacttagg cagcttgatg
     9781 atcaggtcct ttttcacttc tttgtagccc ttggcttcca gaaagtcgat gggattcttc
     9841 tcgaagctgc ttctttccat gatggtgatc cccagcagct ctttcacact cttcagtttc
     9901 ttggacttgc ccttttccac tttggccacc accagcacag aataggccac ggtggggctg
     9961 tcgaagccgc cgtacttctt agggtcccag tccttctttc tggcgatcag cttatcgctg
    10021 ttcctcttgg gcaggataga ctctttgctg aagccgcctg tctgcacctc ggtctttttc
    10081 acgatattca cttggggcat gctcagcact ttccgcacgg tggcaaaatc ccggccctta
    10141 tcccacacga tctccccggt ttcgccgttt gtctcgatca gaggccgctt ccggatctcg
    10201 ccgttggcca gggtaatctc ggtcttgaaa aagttcatga tgttgctgta gaagaagtac
    10261 ttggcggtag ccttgccgat ttcctgctcg ctcttggcga tcatcttccg cacgtcgtac
    10321 accttgtagt cgccgtacac gaactcgctt tccagcttag ggtacttttt gatcagggcg
    10381 gttcccacga cggcgttcag gtaggcgtcg tgggcgtggt ggtagttgtt gatctcgcgc
    10441 actttgtaaa actggaaatc cttccggaaa tcggacacca gcttggactt cagggtgatc
    10501 actttcactt cccggatcag cttgtcattc tcgtcgtact tagtgttcat ccgggagtcc
    10561 aggatctgtg ccacgtgctt tgtgatctgc cgggtttcca ccagctgtct cttgatgaag
    10621 ccggccttat ccagttcgct caggccgcct ctctcggcct tggtcagatt gtcgaacttt
    10681 ctctgggtaa tcagcttggc gttcagcagc tgccgccagt agttcttcat cttcttcacg
    10741 acctcttcgg agggcacgtt gtcgctcttg ccccggttct tgtcgcttct ggtcagcacc
    10801 ttgttgtcga tggagtcgtc cttcagaaag ctctgaggca cgatatggtc cacatcgtag
    10861 tcggacagcc ggttgatgtc cagttcctgg tccacgtaca tatcccgccc attctgcagg
    10921 tagtacaggt acagcttctc gttctgcagc tgggtgtttt ccacggggtg ttctttcagg
    10981 atctggctgc ccagctcttt gatgccctct tcgatccgct tcattctctc gcggctgttc
    11041 ttctgtccct tctgggtggt ctggttctct ctggccattt cgatcacgat gttctcgggc
    11101 ttgtgccggc ccatcacttt cacgagctcg tccaccacct tcactgtctg caggatgccc
    11161 ttcttaatgg cggggctgcc ggccagattg gcaatgtgct cgtgcaggct atcgccctgg
    11221 ccggacacct gggctttctg gatgtcctct ttaaaggtca ggctgtcgtc gtggatcagc
    11281 tgcatgaagt ttctgttggc gaagccgtcg gacttcagga aatccaggat tgtcttgccg
    11341 gactgcttgt cccggatgcc gttgatcagc ttccggctca gcctgcccca gccggtgtat
    11401 ctccgccgct tcagctgctt catcactttg tcgtcgaaca ggtgggcata ggttttcagc
    11461 cgttcctcga tcatctctct gtcctcaaac agtgtcaggg tcagcacgat atcttccaga
    11521 atgtcctcgt tttcctcatt gtccaggaag tccttgtcct tgataatttt cagcagatcg
    11581 tggtatgtgc ccagggaggc gttgaaccga tcttccacgc cggagatttc cacggagtcg
    11641 aagcactcga ttttcttgaa gtagtcctct ttcagctgct tcacggtcac tttccggttg
    11701 gtcttgaaca gcaggtccac gatggccttt ttctgctcgc cgctcaggaa ggcgggcttt
    11761 ctcattccct cggtcacgta tttcactttg gtcagctcgt tatacacggt gaagtactcg
    11821 tacagcaggc tgtgcttggg cagcaccttc tcgttgggca ggttcttatc gaagttggtc
    11881 atccgctcga tgaagctctg ggcggaagcg cccttgtcca ccacttcctc gaagttccag
    11941 ggggtgatgg tttcctcgct ctttctggtc atccaggcga atctgctgtt tcccctggcc
    12001 agagggccca cgtagtaggg gatgcggaag gtcaggatct tctcgatctt ttcccggttg
    12061 tccttcagga atgggtaaaa atcttcctgc cgccgcagaa tggcgtgcag ctctcccagg
    12121 tggatctggt gggggatgct gccgttgtcg aaggtccgct gcttccgcag caggtcctct
    12181 ctgttcagct tcacgagcag ttcctcggtg ccgtccatct tttccaggat gggcttgatg
    12241 aacttgtaga actcttcctg gctggctccg ccgtcaatgt agccggcgta gccgttcttg
    12301 ctctggtcga agaaaatctc tttgtacttc tcaggcagct gctgccgcac gagagctttc
    12361 agcagggtca ggtcctggtg gtgctcgtcg tatctcttga tcatagaggc gctcaggggg
    12421 gccttggtga tctcggtgtt cactctcagg atgtcgctca gcaggatggc gtcggacagg
    12481 ttcttggcgg ccagaaacag gtcggcgtac tggtcgccga tctgggccag caggttgtcc
    12541 aggtcgtcgt cgtaggtgtc cttgctcagc tgcagtttgg catcctcggc caggtcgaag
    12601 ttgctcttga agttgggggt caggcccagg ctcagggcaa tcaggtttcc gaacaggcca
    12661 ttcttcttct cgccgggcag ctgggcgatc agattttcca gccgtctgct cttgctcagt
    12721 ctggcagaca ggatggcctt ggcgtccacg ccgctggcgt tgatggggtt ttcctcgaac
    12781 agctggttgt aggtctgcac cagctggatg aacagcttgt ccacgtcgct gttgtcgggg
    12841 ttcaggtcgc cctcgatcag gaagtggccc cggaacttga tcatgtgggc cagggccaga
    12901 tagatcagcc gcaggtcggc cttgtcggtg ctgtccacca gtttctttct caggtggtag
    12961 atggtggggt acttctcgtg gtaggccacc tcgtccacga tgttgccgaa gatggggtgc
    13021 cgctcgtgct tcttatcctc ttccaccagg aaggactctt ccagtctgtg gaagaagctg
    13081 tcgtccacct tggccatctc gttgctgaag atctcttgca gatagcagat ccggttcttc
    13141 cgtctggtgt atcttcttct ggcggttctc ttcagccggg tggcctcggc tgtttcgccg
    13201 ctgtcgaaca gcagggctcc gatcaggttc ttcttgatgc tgtgccggtc ggtgttgccc
    13261 agcaccttga atttcttgct gggcaccttg tactcgtcgg tgatcacggc ccagcccaca
    13321 gagttggtgc cgatgtccag gccgatgctg tacttcttgt cggctgctgg gactccgtgg
    13381 ataccgacct tccgcttctt ctttggggcc atcttatcgt catcgtcttt gtaatcaata
    13441 tcatgatcct tgtagtctcc gtcgtggtcc ttatagtcca tggtggcacc ggtcctgtgt
    13501 tctggcggca aacccgttgc gaaaaagaac gttcacggcg actactgcac ttatatacgg
    13561 ttctccccca ccctcgggaa aaaggcggag ccagtacacg acatcacttt cccagtttac
    13621 cccgcgccac cttctctagg caccggatca attgccgacc cctcccccca acttctcggg
    13681 gactgtgggc gatgtgcgct ctgcccactg acgggcaccg gagccaattc ccactccttt
    13741 caagacctag ctagattgta aaaaacaagt tgataacgga ctagccttca acaatctaat
    13801 aaaccatact ttcatggttt attaattacg agttcaagag gcctggatga cagtggtctc
    13861 atacagaact tataagattc ccaaatccaa agacatttca cgtttatggt gatttcccag
    13921 aacacatagc gacatgcaaa tattgcaggg cgccactccc ctgtccctca cagccatctt
    13981 cctgccaggg cgcacgcgcg ctgggtgttc ccgcctagtg acactgggcc cgcgattcct
    14041 tggagcgggt tgatgacgtc agcgttcctc gagaaaaaac aagttgataa cggactagcc
    14101 ttcaacaatc taataaacca tactttcatg gtttattaat tacgagaggt ggac
//
