pRDA_550

LOCUS        pRDA_550       12301 bp    DNA     circular SYN 14-APR-2025
DEFINITION  All-in-one lentiviral expression of gRNA and EnAsCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12301)
  AUTHORS   Esmaeili Anvar N, Lin C, Ma X, Wilson LL, Steger R, Sangree AK, 
            Colic M, Wang SH, Doench JG, Hart T
  TITLE     Efficient gene knockout and genetic interaction screening using the 
            in4mer CRISPR/Cas12a multiplex knockout platform.
  JOURNAL   Nat Commun. 2024 Apr 27;15(1):3577. doi: 10.1038/s41467-024-47795-3.
  PUBMED    38678031
REFERENCE   2  (bases 1 to 12301)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.biofeng.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2024-04-27"; volume: "15(1):3577. doi"; pages: " 
            10.1038/s41467-024-47795-3"
COMMENT     Sequence Label: pRDA_550
FEATURES             Location/Qualifiers
     source          1..12301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..27)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10..26)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(19..41)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(235..254)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     354..376
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(414..432)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        500..604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(605..673,674..1465)
                     /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(823..842)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1636..2224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2378..2395
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2512..2533
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2548..2553,2554..2571,2572..2578)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2586..2602
                     /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     2591..2613
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2610..2626
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2610..2626
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      2659..2794
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2834..2955
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2845..2864)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2899..2918
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             3027..3260
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    3334..3922
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3440..3451
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     3849..3869
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3932..4528)
                     /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(4015..4035)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             complement(4535..4588)
                     /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(4604..4684)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(4691..4738)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(4739..8656)
                     /codon_start=1
                     /product="CRISPR-associated protein from Acidaminococcus 
                     sp. BV3L6 (Zetsche et al., 2015)"
                     /label=AsCpf1
                     /translation="TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH
                     YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNA
                     IHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDK
                     FTTYFSGFYRNRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE
                     NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLN
                     LAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLR
                     NENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGK
                     ITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLK
                     KQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKAR
                     NYATKKPYSVEKFKLNFQMPTLARGWDVNREKNNGAILFVKNGLYYLGIMPKQKGRYKA
                     LSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPL
                     EITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDL
                     SSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH
                     HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKD
                     QKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFH
                     VPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRS
                     LNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVV
                     VLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQF
                     TSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDV
                     KTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENH
                     RFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMR
                     NSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESK
                     DLKLQNGISNQDWLAYIQELRN"
     regulatory      5010..5019
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(8681..8701)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      8704..8713
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(8722..9899)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          8731..9668
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(8754..8774)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        9992..10240
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     9992..10012
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     10163..10182
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    10369..10484
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     CDS             complement(10762..10806)
                     /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    10991..11224
                     /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    11717..11842
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             11889..12069
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(12070..12296)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
ORIGIN
        1 cagcttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
       61 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
      121 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
      181 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
      241 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
      301 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg actccgggag
      361 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagtcgaa agggcctcgt
      421 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
      481 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
      541 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
      601 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
      661 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
      721 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
      781 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
      841 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
      901 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
      961 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     1021 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     1081 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     1141 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     1201 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     1261 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     1321 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     1381 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     1441 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     1501 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1561 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1621 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1681 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1741 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     1801 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1861 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1921 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1981 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2041 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2101 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2161 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     2221 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2281 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2341 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2401 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     2461 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     2521 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     2581 tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga ttacgaattt
     2641 ggtctctgca aaagtctagg cctccaaaaa agcctcctca ctacttctgg aatagctcag
     2701 aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca tggggcggag
     2761 aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc gggatagcta
     2821 gagccagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     2881 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     2941 tgcaataaac aagttcctct cactctctga tattcatttc tttgcaagtt ataaatactg
     3001 aataataaga tgacatgaac tactactgct agagattttc cacactgact aaaagggtct
     3061 gagggatctc tagttaccag agtcacacaa cagacgggca cacactactt gaagcactca
     3121 aggcaagctt tattgaggct taagcagtgg gttccctagt tagccagaga gctcccaggc
     3181 tcagatctgg tctaaccaga gagacccagt acaagcaaaa agcagatctt atcttcgttg
     3241 ggagtgaatt agcccttcca gtcccccctt ttcttttaaa aagtggctaa gatctacagc
     3301 tgccttgtaa gtcattggtc ttaaaggtac caggcgggga ggcggcccaa agagagatcc
     3361 gactcgtctg agggcgaagg cgaggacgcg gaagaggccg cagagccggc agcaggccgc
     3421 gggaaggaag gtccgctgga ttgagggccg aagggacgta gcagaaggac gtcccgcgca
     3481 gaatccaggt ggcaacacag gcgagcagcc aaggaaagga cgatgatttc cccgacaaca
     3541 ccacggaatt gtcagtgccc aacagccgag cccctgtcca gcagcgggca aggcaggcgg
     3601 cgatgagttc cgccgtggca atagggaggg ggaaagcgaa agtcccggaa aggagctgac
     3661 aggtggtggc aatgccccaa ccagtggggg ttgcgtcagc aaacacagtg cacaccacgc
     3721 cacgttgcct gacaacgggc cacaactcct cataaagaga cagcaaccag gatttataca
     3781 aggaggagaa aatgaaagcc atacgggaag caatagcatg atacaaaggc attaaagcag
     3841 cgtatccaca tagcgtaaaa ggagcaacat agttaagaat accagtcaat ctttcacaaa
     3901 ttttgtaatc cagaggttga ttgtcgactt atcaggcacc gggcttgcgg gtcatgcacc
     3961 aggtgcgcgg tccttcgggc acctcgacgt cggcggtgac ggtgaagccg agccgctcgt
     4021 agaaggggag gttgcggggc gcggaggtct ccaggaaggc gggcaccccg gcgcgctcgg
     4081 ccgcctccac tccggggagc acgacggcgc tgcccagacc cttgccctgg tggtcgggcg
     4141 agactccgac ggtggccagg aaccacgcgg gctccttggg ccggtgcggc gccaggaggc
     4201 cttccatctg ttgctgcgcg gccagccggg aaccgctcaa ctcggccatg cgcgggccga
     4261 tctcggcgaa caccgccccc gcttcgacgc tctccggcgt ggtccagacc gccaccgcgg
     4321 cgccgtcgtc cgcgacccac accttgccga tgtcgagccc gacgcgcgtg aggaagagtt
     4381 cttgcagctc ggtgacccgc tcgatgtggc ggtccggatc gacggtgtgg cgcgtggcgg
     4441 ggtagtcggc gaacgcggcg gcgagggtgc gtacggccct ggggacgtcg tcgcgggtgg
     4501 cgaggcgcac cgtgggcttg tactcggtcc cggaagggcc gggattctcc tccacgtcac
     4561 cgcatgttag aaggcttcct ctgccctcgc atcctgcggc cgcggcatag tcggggacat
     4621 catatgggta tgcataatca ggcacgtcgt agggataagc gtaatctgga acatcgtatg
     4681 ggtaggatcc ctttttcttt tttgcctggc cggccttttt cgtggccgcc ggccttttgt
     4741 tgcgcagctc ctggatgtag gccagccagt cctgattgga gatgccgttc tgcagcttca
     4801 gatccttgct ctccttcagg tgattcagca gcagctggcc cttcagggcg atgtggtagg
     4861 cgccattggc atcggcgtcc atgggccact ctgggttctg aaaccgggag tcgaagcaca
     4921 cgccattcag atcgcgcacg gggctgttga tatagtcctc gcctgtggcg gcattggagt
     4981 tccgcatctg cagcacgctg cggatcaggg ccaccatggt gtcgatggcg tgagaatcgt
     5041 cattctccag cagctttggc aggatgttgg agccatccct gaacacgatg cccttctcct
     5101 ccagcagggc gatcagctcg ttggcaggat acaggtcccg gtatctgccg gtgaatctgt
     5161 gattctcgat cactggcacg attctcttgc cggcgatgaa aggggtgccc ttggcgtcaa
     5221 actgtgtctc gttcttctcg aacacgatat cccatgcagg cataaagccg ggcaggcccc
     5281 tctggaagga cagatttctg ttcatcttaa agtgcaggat gaagtcgccg gttttcacgt
     5341 cgtagtgcag aaagtcgaag ccctccagga agtgcttgcg gctctcgtga ttcttgatgg
     5401 ttttccacac gaaggggtcc acgaagccgg tcaggggatc gatcttagat gtatatgggg
     5461 caggcacgta aaacaggaag ccagactggg tgcccatctt ggcaaaggag gtgaactggt
     5521 ctgtcagctg gtatgggttc agcacgcctc ccactttctc tgctggatag tccttcagca
     5581 ccaggcaatt cagcttatcg atcagcatct tctcgaactg ctggtacacg gccttctcgg
     5641 cgatgccggt cctcttgctc ttaaagccga aattcaggtt ctccagcacc accacggcct
     5701 ggtagtggat catcaggtcc acgatctcgt ggatgacctg gctcagatag ccctgcttca
     5761 gatccttgat tgtgcccacc acagaccagg cctgccttgc tgccaccctc tccttctccc
     5821 tgttgtccag cttcttctgg taatcaaact gctggatggt gttcaggctc cgctgctcca
     5881 ggatcttgcc ggtggagtcg atcactgtga tatagatcag gtttctctcg ccccgatcga
     5941 tgccgatgat aggtgtctcg gggtgctcct tcaggtaggc attcaccctc tggttgaact
     6001 tagatgggga attggcggcc tgatagttca gtgtgatagg cacgtggaaa aagaacttgt
     6061 cgctggtaaa gcgcctatcc ttgatgatct cgtgagacac ctccttggtg atcacgttgg
     6121 gcagcagggc cctggcctca tcagacaggt cgtgggacag tctgtgattc acatagtcgt
     6181 acagctcctg gtacagggtg tcggggattg gggttttctg atccttcagc ttcttgttca
     6241 gcatcttctc tcccagccgg tgtgccatcc tcttcatcct ggacttaggg cggtagaaca
     6301 gctcggcctg gccattcagc ttgatgcttg tcttggccag gttctctgga gaaaacaggc
     6361 cggtccaata cagtgtgtgc agattaggct tgccgtggtg gcccttggca aagtccttgt
     6421 tatagatctg gaacaggtac agcttgcctg tctccacggc atccatgatc tccttctcgg
     6481 cgattctctg gaagctgatg tggtacagca ggggattcag ctcggcatag tactcgccca
     6541 ggtccttata ctgagaggat ggccgcaggc tagacagatc gatagaggtt gtcttggtat
     6601 acttggacag aaaatccctt gtgaagtcga tccacttgca cagggcctct ctgtagccct
     6661 tctggtcgcc ggttttcttg gcgtaggctg tctgaaactt ctttggctcc ttctcaggat
     6721 tgttcaggtc gtagatctcc tttgtgatct ccagaggctc gatgaaattg ttggacagca
     6781 ggatgggggt tgtgtgggtc tgaaagtggg ctgtcacggc cttcagctgg gtgctgcact
     6841 ttgggatcat cttggcggca tcagggaagt agtcatagta catcttatca aagccctcgc
     6901 tggttttctc tgtgggctcg aagctcaggg ccttatacct gcccttctgc tttggcatga
     6961 tgcccagata gtacaggccg ttcttcacaa acaggatggc gccattgttc ttctcccgat
     7021 tcacgtccca gccgcgggcc agtgtaggca tctgaaagtt cagcttgaac ttctccacgg
     7081 agtagggctt cttggtggca taatttctgg ccttgttgta gaagctcaga gaaggctcca
     7141 tctccagctt gatgccggtc agccgggcag agaactcggg gtccacctcg ttggactcat
     7201 ccacggcaaa ccagtccagc aggtggtaca ggcccagcag gctgtccagc tgagacttca
     7261 ggatctcctt ctcctcctgc ttcttcaggg ttgtaggcag tggctgatcc agggcggcgt
     7321 gtgcgtggga caggatctcg ctggttttct gcttgaaggc ctcgctcagc tccttgcctg
     7381 cggcagagat gatctcctgc aggttgatat cctcgtgctt caggctgcgc tgcaccttct
     7441 ccttggcaga cttggtgatc ttgcctgtca gctcggagat tctccgctca tacagggcat
     7501 tcctcagtgt atcccagtgg tcgcacaggg cgctgctgat tgtctccagc ttcttgtggc
     7561 tgatgaagat gtgtgtcagg tcgatgctgt tcagctcgtt aaacagggcc tcggctgtct
     7621 ccagcacgtt ctcgtttctc agcagtgtct tgtacttgca gaaggactgg atcacttcct
     7681 cgtcgctctt aaactcctcc aggatgaaag acagggtgtt cctatcggac aggatctgct
     7741 taaacagggg gatgaatctg tgtggcaggg aggcgatgat gtgggctgtc tcatcattct
     7801 tctggatggc cagattcagc acctcgttca ggcccttgat cttctcggtg cctgcctccc
     7861 gagagattcc tcccagcagc tggttataca ggtcgatctg ggtctgtgtc agcagctggt
     7921 tataaaaagg gaaggaaaac acctcctcga tggaggtgct cacgaagatg ccgatggcct
     7981 tcttcacgtt ctcaaagtgc tcccgcaggc tgggcacggc ggtgatcagg cgtgtgaaga
     8041 tgtgacaatt ctccttaaac ttggggaagt tgtcctgcac gatgcggtgt gggatggctg
     8101 tgctgatatc ctcggcgctg aacacgttct tcctgttccg ataaaagccg gagaagtagg
     8161 ttgtaaactt gtcgaagctc cgcagcaggg cgttctcgtg ctcggttgtg gtcacggtgc
     8221 ccagctgctt cagcaccttg ccattaaaca gctcggcctt gaacaggccc ttgtagatct
     8281 cggcgtgtct cttattgatg gcatcggtca ggttgtctgt ccggccgatg aagtagtcgt
     8341 ggatggcatt gcgatatgtg gcctgctcct cgatcagggc gttccttgtc tcctcggttt
     8401 tctcctttct ataggagtcg atggcggcgc tcaggttctc ccaatccagc tgcaccagct
     8461 gcaggcactg gtcggcatag gtcttgtaga tccgatcgat gatgggcttc agctccttgt
     8521 agtgatcatt gcgggccttg tcctcctcga tgaagccctg ctcctggatg tgcttcaggg
     8581 tcttgccctg tgggatcagc tcaaaccgca gtgtcttgct cacctgatac aggttggtaa
     8641 agccctcgaa ctgtgtggct gctgggactc cgtggatacc gaccttccgc ttcttctttg
     8701 gggccatggt ggcggctaga atcacgacac ctgaaatgga agaaaaaaac tttgaaccac
     8761 tgtctgaggc ttgagaatga accaagatcc aaactcaaaa agggcaaatt ccaaggagaa
     8821 ttacatcaag tgccaagctg gcctaacttc agtctccacc cactcagtgt ggggaaactc
     8881 catcgcataa aacccctccc cccaacctaa agacgacgta ctccaaaagc tcgagaacta
     8941 atcgaggtgc ctggacggcg cccggtactc cgtggagtca catgaagcga cggctgagga
     9001 cggaaaggcc cttttccttt gtgtgggtga ctcacccgcc cgctctcccg agcgccgcgt
     9061 cctccatttt gagctccctg cagcagggcc gggaagcggc catctttccg ctcacgcaac
     9121 tggtgccgac cgggccagcc ttgccgccca gggcggggcg atacacggcg gcgcgaggcc
     9181 aggcaccaga gcaggccggc cagcttgaga ctacccccgt ccgattctcg gtggccgcgc
     9241 tcgcaggccc cgcctcgccg aacatgtgcg ctgggacgca cgggccccgt cgccgcccgc
     9301 ggccccaaaa accgaaatac cagtgtgcag atcttggccc gcatttacaa gactatcttg
     9361 ccagaaaaaa agcgtcgcag caggtcatca aaaattttaa atggctagag acttatcgaa
     9421 agcagcgaga caggcgcgaa ggtgccacca gattcgcacg cggcggcccc agcgcccagg
     9481 ccaggcctca actcaagcac gaggcgaagg ggctccttaa gcgcaaggcc tcgaactctc
     9541 ccacccactt ccaacccgaa gctcgggatc aagaatcacg tactgcagcc agtggaagta
     9601 attcaaggca cgcaagggcc ataacccgta aagaggccag gcccgcggga accacacacg
     9661 gcacttacct gtgttctggc ggcaaacccg ttgcgaaaaa gaacgttcac ggcgactact
     9721 gcacttatat acggttctcc cccaccctcg ggaaaaaggc ggagccagta cacgacatca
     9781 ctttcccagt ttaccccgcg ccaccttctc taggcaccgg ttcaattgcc gacccctccc
     9841 cccaacttct cggggactgt gggcgatgtg cgctctgccc actgacgggc accggagcca
     9901 attcccactc ctttcaagac ctagaaggtc cattagctgc aaagattcct ctctgtttaa
     9961 aactttatcc atctttgcaa ctagattcga agagggccta tttcccatga ttccttcata
    10021 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
    10081 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
    10141 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
    10201 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggtaatttct actcttgtag
    10261 atggagacgc gttttttctc gtctctgaat ctggtcttga aaaagtggca ccgagtcggt
    10321 gaattcagta gcgtgaagag cagagaacga gcaccgacaa caacgcagaa attttgaatt
    10381 tttgtaattt gtttttgtaa ttctttagtt tgtatgtctg ttgctattat gtctactatt
    10441 ctttcccctg cactgtaccc cccaatcccc ccttttcttt taaattgctc gatccaagca
    10501 acgataccgt cgagatccgt tcactaatcg aatggatctg tctctgtctc tctctccacc
    10561 ttcttcttct attccttcgg gcctgtcggg tcccctcggg gttgggaggt gggtctgaaa
    10621 cgataatggt gaatatccct gcctaactct attcactata gaaagtacag caaaaactat
    10681 tcttaaacct accaagcctc ctactatcat tatgaataat tttatatacc acagccaatt
    10741 tgttatgtta aaccaattcc acaaacttgc ccatttatct aattccaata attcttgttc
    10801 attcttttct tgctggtttt gcgattcttc aattaaggag tgtattaagc ttgtgtaatt
    10861 gttaatttct ctgtcccact ccatccaggt cgtgtgattc caaatctgtt ccagagattt
    10921 attactccaa ctagcattcc aaggcacagc agtggtgcaa atgagttttc cagagcaacc
    10981 ccaaatcccc aggagctgtt gatcctttag gtatctttcc acagccagga ttcttgcctg
    11041 gagctgcttg atgccccaga ctgtgagttg caacagatgc tgttgcgcct caatagccct
    11101 cagcaaattg ttctgctgct gcactatacc agacaataat tgtctggcct gtaccgtcag
    11161 cgtcattgac gctgcgccca tagtgcttcc tgctgctccc aagaacccaa ggaacaaagc
    11221 tcctattccc actgctcttt tttctctctg caccactctt ctctttgcct tggtgggtgc
    11281 tactcctaat ggttcaattt ttactacttt atatttatat aattcacttc tccaattgtc
    11341 cctcatatct cctcctccag gtctgaagat cagtggccgc ttgctgtgcg gtggtcttac
    11401 ttttgttttg ctcttcctct atcttgtcta aagcttcctt ggtgtctttt atctctatcc
    11461 tttgatgcac acaatagagg gttgctactg tattatataa tgatctaagt tcttctgatc
    11521 ctgtctgaag ggatggttgt agctgtccca gtatttgtct acagccttct gatgtttcta
    11581 acaggccagg attaactgcg aatcgttcta gctccctgct tgcccatact atatgtttta
    11641 atttatattt tttctttccc cctggcctta accgaatttt ttcccatcgc gatctaattc
    11701 tcccccgctt aatactgacg ctctcgcacc catctctctc cttctagcct ccgctagtca
    11761 aaatttttgg cgtactcacc agtcgccgcc cctcgcctct tgccgtgcgc gcttcagcaa
    11821 gccgagtcct gcgtcgagag agctcctctg gtttcccttt cgctttcaag tccctgttcg
    11881 ggcgccactg ctagagattt tccacactga ctaaaagggt ctgagggatc tctagttacc
    11941 agagtcacac aacagacggg cacacactac ttgaagcact caaggcaagc tttattgagg
    12001 cttaagcagt gggttcccta gttagccaga gagctcccag gctcagatct ggtctaacca
    12061 gagagacccg tttatgtatc gagctaggca cttaaataca atatctctgc aatgcggcaa
    12121 ttcagtggtt cgtccaatcc atgtcagacc cgtctgttgc cttcctaata aggcacgatc
    12181 gtaccacctt acttccacca atcggcatgc acggtgcttt ttctctcctt gtaaggcatg
    12241 ttgctaactc atcgttacca tgttgcaaga ctacaagagt attgcataag actacacgcg
    12301 t
//