LOCUS       sequence-263333-       11785 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Lentiviral expression of AsCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11785)
  AUTHORS   DeWeirdt PC, Sanson KR, Sangree AK, Hegde M, Hanna RE, Feeley MN, 
            Griffith AL, Teng T, Borys SM, Strand C, Joung JK, Kleinstiver BP, 
            Pan X, Huang A, Doench JG
  TITLE     Optimization of AsCas12a for combinatorial genetic screens in human 
            cells.
  JOURNAL   Nat Biotechnol. 2020 Jul 13. pii: 10.1038/s41587-020-0600-6. doi: 
            10.1038/s41587-020-0600-6.
  PUBMED    32661438
REFERENCE   2  (bases 1 to 11785)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2020 Jul 13. pii: 10.1038/s41587-020-0600-6. doi: 
            10.1038/s41587-020-0600-6."
COMMENT     Sequence Label: pRDA_112
FEATURES             Location/Qualifiers
     source          1..11785
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..232
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             233..413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /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    1818..1933
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        2085..3262
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2316..3253
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3210..3230
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3271..3280
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3283..3303
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3328..7245
                     /codon_start=1
                     /product="CRISPR-associated protein from Acidaminococcus 
                     sp. BV3L6 (Zetsche et al., 2015)"
                     /label=AsCpf1
                     /translation="TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH
                     YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNA
                     IHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDK
                     FTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE
                     NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLN
                     LAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLR
                     NENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGK
                     ITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLK
                     KQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKAR
                     NYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKA
                     LSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPL
                     EITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDL
                     SSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH
                     HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKD
                     QKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFH
                     VPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRS
                     LNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVV
                     VLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQF
                     TSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDV
                     KTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENH
                     RFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMR
                     NSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESK
                     DLKLQNGISNQDWLAYIQELRN"
     regulatory      6965..6974
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7246..7293
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7300..7380
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             7396..7449
                     /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             7456..7854
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    7864..8452
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7917..7937)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8335..8346)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8526..8759
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8831..8952
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8868..8887)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8922..8941
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8992..9127
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9160..9176)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9160..9176)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9173..9195)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9184..9200
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(9208..9214,9215..9232,9233..9238))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9253..9274
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9391..9408)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9562..10150)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(10321..11112,11113..11181))
                     /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     10944..10963
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11182..11286)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     11354..11372
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(11410..11432)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     11532..11551
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     11745..11767
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     11759..11776
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     11760..11776
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      241 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      301 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      361 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      421 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccact gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
     1741 aggtggagag agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg
     1801 ttgcttggat cgagcaattt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa
     1861 gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa
     1921 aaattcaaaa tttctgcgtt gttgtcggtg ctcgttctct gctcttcacg ctactgaatt
     1981 catcaccggt tcttcgaatc tagttgcaaa gatggataaa gttttaaaca gagaggaatc
     2041 tttgcagcta atggaccttc taggtcttga aaggagtggg aattggctcc ggtgcccgtc
     2101 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
     2161 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     2221 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     2281 ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg
     2341 ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc actggctgca
     2401 gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg
     2461 cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg
     2521 ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc
     2581 catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa
     2641 tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc
     2701 ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat
     2761 cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg
     2821 tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag
     2881 atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga
     2941 gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc
     3001 atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg
     3061 gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga
     3121 gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc
     3181 cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt
     3241 tcttccattt caggtgtcgt gattctagcc gccaccatgg ccccaaagaa gaagcggaag
     3301 gtcggtatcc acggagtccc agcagccaca cagttcgagg gctttaccaa cctgtatcag
     3361 gtgagcaaga cactgcggtt tgagctgatc ccacagggca agaccctgaa gcacatccag
     3421 gagcagggct tcatcgagga ggacaaggcc cgcaatgatc actacaagga gctgaagccc
     3481 atcatcgatc ggatctacaa gacctatgcc gaccagtgcc tgcagctggt gcagctggat
     3541 tgggagaacc tgagcgccgc catcgactcc tatagaaagg agaaaaccga ggagacaagg
     3601 aacgccctga tcgaggagca ggccacatat cgcaatgcca tccacgacta cttcatcggc
     3661 cggacagaca acctgaccga tgccatcaat aagagacacg ccgagatcta caagggcctg
     3721 ttcaaggccg agctgtttaa tggcaaggtg ctgaagcagc tgggcaccgt gaccacaacc
     3781 gagcacgaga acgccctgct gcggagcttc gacaagttta caacctactt ctccggcttt
     3841 tatgagaaca ggaagaacgt gttcagcgcc gaggatatca gcacagccat cccacaccgc
     3901 atcgtgcagg acaacttccc caagtttaag gagaattgtc acatcttcac acgcctgatc
     3961 accgccgtgc ccagcctgcg ggagcacttt gagaacgtga agaaggccat cggcatcttc
     4021 gtgagcacct ccatcgagga ggtgttttcc ttcccttttt ataaccagct gctgacacag
     4081 acccagatcg acctgtataa ccagctgctg ggaggaatct ctcgggaggc aggcaccgag
     4141 aagatcaagg gcctgaacga ggtgctgaat ctggccatcc agaagaatga tgagacagcc
     4201 cacatcatcg cctccctgcc acacagattc atccccctgt ttaagcagat cctgtccgat
     4261 aggaacaccc tgtctttcat cctggaggag tttaagagcg acgaggaagt gatccagtcc
     4321 ttctgcaagt acaagacact gctgagaaac gagaacgtgc tggagacagc cgaggccctg
     4381 tttaacgagc tgaacagcat cgacctgaca cacatcttca tcagccacaa gaagctggag
     4441 acaatcagca gcgccctgtg cgaccactgg gatacactga ggaatgccct gtatgagcgg
     4501 agaatctccg agctgacagg caagatcacc aagtctgcca aggagaaggt gcagcgcagc
     4561 ctgaagcacg aggatatcaa cctgcaggag atcatctctg ccgcaggcaa ggagctgagc
     4621 gaggccttca agcagaaaac cagcgagatc ctgtcccacg cacacgccgc cctggatcag
     4681 ccactgccta caaccctgaa gaagcaggag gagaaggaga tcctgaagtc tcagctggac
     4741 agcctgctgg gcctgtacca cctgctggac tggtttgccg tggatgagtc caacgaggtg
     4801 gaccccgagt tctctgcccg gctgaccggc atcaagctgg agatggagcc ttctctgagc
     4861 ttctacaaca aggccagaaa ttatgccacc aagaagccct actccgtgga gaagttcaag
     4921 ctgaactttc agatgcctac actggcctct ggctgggacg tgaataagga gaagaacaat
     4981 ggcgccatcc tgtttgtgaa gaacggcctg tactatctgg gcatcatgcc aaagcagaag
     5041 ggcaggtata aggccctgag cttcgagccc acagagaaaa ccagcgaggg ctttgataag
     5101 atgtactatg actacttccc tgatgccgcc aagatgatcc caaagtgcag cacccagctg
     5161 aaggccgtga cagcccactt tcagacccac acaaccccca tcctgctgtc caacaatttc
     5221 atcgagcctc tggagatcac aaaggagatc tacgacctga acaatcctga gaaggagcca
     5281 aagaagtttc agacagccta cgccaagaaa accggcgacc agaagggcta cagagaggcc
     5341 ctgtgcaagt ggatcgactt cacaagggat tttctgtcca agtataccaa gacaacctct
     5401 atcgatctgt ctagcctgcg gccatcctct cagtataagg acctgggcga gtactatgcc
     5461 gagctgaatc ccctgctgta ccacatcagc ttccagagaa tcgccgagaa ggagatcatg
     5521 gatgccgtgg agacaggcaa gctgtacctg ttccagatct ataacaagga ctttgccaag
     5581 ggccaccacg gcaagcctaa tctgcacaca ctgtattgga ccggcctgtt ttctccagag
     5641 aacctggcca agacaagcat caagctgaat ggccaggccg agctgttcta ccgccctaag
     5701 tccaggatga agaggatggc acaccggctg ggagagaaga tgctgaacaa gaagctgaag
     5761 gatcagaaaa ccccaatccc cgacaccctg taccaggagc tgtacgacta tgtgaatcac
     5821 agactgtccc acgacctgtc tgatgaggcc agggccctgc tgcccaacgt gatcaccaag
     5881 gaggtgtctc acgagatcat caaggatagg cgctttacca gcgacaagtt ctttttccac
     5941 gtgcctatca cactgaacta tcaggccgcc aattccccat ctaagttcaa ccagagggtg
     6001 aatgcctacc tgaaggagca ccccgagaca cctatcatcg gcatcgatcg gggcgagaga
     6061 aacctgatct atatcacagt gatcgactcc accggcaaga tcctggagca gcggagcctg
     6121 aacaccatcc agcagtttga ttaccagaag aagctggaca acagggagaa ggagagggtg
     6181 gcagcaaggc aggcctggtc tgtggtgggc acaatcaagg atctgaagca gggctatctg
     6241 agccaggtca tccacgagat cgtggacctg atgatccact accaggccgt ggtggtgctg
     6301 gagaacctga atttcggctt taagagcaag aggaccggca tcgccgagaa ggccgtgtac
     6361 cagcagttcg agaagatgct gatcgataag ctgaattgcc tggtgctgaa ggactatcca
     6421 gcagagaaag tgggaggcgt gctgaaccca taccagctga cagaccagtt cacctccttt
     6481 gccaagatgg gcacccagtc tggcttcctg ttttacgtgc ctgccccata tacatctaag
     6541 atcgatcccc tgaccggctt cgtggacccc ttcgtgtgga aaaccatcaa gaatcacgag
     6601 agccgcaagc acttcctgga gggcttcgac tttctgcact acgacgtgaa aaccggcgac
     6661 ttcatcctgc actttaagat gaacagaaat ctgtccttcc agaggggcct gcccggcttt
     6721 atgcctgcat gggatatcgt gttcgagaag aacgagacac agtttgacgc caagggcacc
     6781 cctttcatcg ccggcaagag aatcgtgcca gtgatcgaga atcacagatt caccggcaga
     6841 taccgggacc tgtatcctgc caacgagctg atcgccctgc tggaggagaa gggcatcgtg
     6901 ttcagggatg gctccaacat cctgccaaag ctgctggaga atgacgattc tcacgccatc
     6961 gacaccatgg tggccctgat ccgcagcgtg ctgcagatgc ggaactccaa tgccgccaca
     7021 ggcgaggact atatcaacag ccccgtgcgc gatctgaatg gcgtgtgctt cgactcccgg
     7081 tttcagaacc cagagtggcc catggacgcc gatgccaatg gcgcctacca catcgccctg
     7141 aagggccagc tgctgctgaa tcacctgaag gagagcaagg atctgaagct gcagaacggc
     7201 atctccaatc aggactggct ggcctacatc caggagctgc gcaacaaaag gccggcggcc
     7261 acgaaaaagg ccggccaggc aaaaaagaaa aagggatcct acccatacga tgttccagat
     7321 tacgcttatc cctacgacgt gcctgattat gcatacccat atgatgtccc cgactatgcc
     7381 gcggccgcag gatgcgaggg cagaggaagc cttctaacat gcggtgacgt ggaggagaat
     7441 cccggccctt ccgggatggc caagcctttg tctcaagaag aatccaccct cattgaaaga
     7501 gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct
     7561 ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct
     7621 tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt
     7681 atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag
     7741 gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg
     7801 acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaataagtc
     7861 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     7921 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     7981 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     8041 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     8101 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     8161 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     8221 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     8281 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     8341 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     8401 ctccgccttc gccctcagac gagtcggatc tctctttggg ccgcctcccc gcctggtacc
     8461 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     8521 gggactggaa gggctaattc actcccaacg aagataagat ctgctttttg cttgtactgg
     8581 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     8641 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     8701 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     8761 tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga
     8821 gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     8881 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     8941 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc
     9001 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     9061 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     9121 ggaggcctag acttttgcag agaccaaatt cgtaatcatg tcatagctgt ttcctgtgtg
     9181 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     9241 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     9301 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     9361 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     9421 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     9481 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     9541 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     9601 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     9661 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     9721 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     9781 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     9841 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     9901 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     9961 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
    10021 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
    10081 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
    10141 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
    10201 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
    10261 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
    10321 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
    10381 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
    10441 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
    10501 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
    10561 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
    10621 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
    10681 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
    10741 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
    10801 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
    10861 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
    10921 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
    10981 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
    11041 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
    11101 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
    11161 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
    11221 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
    11281 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
    11341 attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga
    11401 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
    11461 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
    11521 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
    11581 accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg
    11641 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
    11701 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
    11761 taaaacgacg gccagtgcca agctg
//
