LOCUS       sequence-370968-       14610 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct encoding doxycycline-inducible AsCas12a (via 
            rtTA3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14610)
  AUTHORS   Hughes NW, Qu Y, Zhang J, Tang W, Pierce J, Wang C, Agrawal A, Morri
            M, Neff N, Winslow MM, Wang M, Cong L
  TITLE     Machine-learning-optimized Cas12a barcoding enables the recovery of 
            single-cell lineages and transcriptional profiles.
  JOURNAL   Mol Cell. 2022 Jun 22. pii: S1097-2765(22)00537-8. doi: 
            10.1016/j.molcel.2022.06.001.
  PUBMED    35752172
REFERENCE   2  (bases 1 to 14610)
  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: "Mol Cell. 
            2022 Jun 22. pii: S1097-2765(22)00537-8. doi: 
            10.1016/j.molcel.2022.06.001."
COMMENT     Sequence Label: pN006
FEATURES             Location/Qualifiers
     source          1..14610
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(98..115)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(269..857)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(349..368)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1028..1819,1820..1888))
                     /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     1651..1670
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1889..1993)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2068..2087)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2259..2638
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2640..2838
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             2856..3036
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3083..3208
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3701..3934
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4119..4163
                     /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    4461..4578
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(4672..4920)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4730..4749)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4900..4920)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     protein_bind    4969..4987
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5005..5023
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5040..5058
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5076..5094
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5112..5130
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5147..5165
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5183..5201
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        5213..5251
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      5264..5273
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5276..5296
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5321..9238
                     /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      8958..8967
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             9239..9286
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9287..9313
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             9338..9391
                     /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             9398..10090
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGH"
     promoter        10116..10327
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             10340..11044
                     /codon_start=1
                     /product="reverse tetracycline transactivator 3"
                     /label=rtTA3
                     /note="activates expression from Tet-responsive promoters 
                     in the presence of doxycycline"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPG"
     CDS             11054..11110
                     /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             11111..11707
                     /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     11604..11624
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    11723..12311
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11776..11796)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12194..12205)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             12383..12616
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(12642..12659)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12648..12872
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      12918..13346
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(13005..13024)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     13215..13236
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(13355..13375)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        13360..13689
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      13540..13675
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     13602..13621
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        13737..13784
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             13803..14177
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    14307..14428
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(14344..14363)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     14398..14417
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(14477..14493)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14477..14493)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(14490..14512)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    14501..14517
                     /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(14525..14531,14532..14549,14550..14555))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14570..14591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
       61 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
      121 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
      181 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
      241 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
      301 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
      361 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
      421 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
      481 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
      541 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
      601 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
      661 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
      721 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
      781 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
      841 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
      901 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
      961 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     1021 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     1081 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     1141 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     1201 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     1261 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     1321 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     1381 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     1441 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     1501 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     1561 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     1621 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     1681 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     1741 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     1801 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     1861 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     1921 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     1981 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     2041 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     2101 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     2161 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     2221 tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta
     2281 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     2341 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     2401 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     2461 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     2521 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     2581 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     2641 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     2701 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     2761 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     2821 ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct
     2881 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     2941 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     3001 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
     3061 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
     3121 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
     3181 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     3241 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     3301 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     3361 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     3421 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     3481 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     3541 gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg
     3601 aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa
     3661 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt
     3721 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca
     3781 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc
     3841 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg
     3901 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac
     3961 tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga
     4021 tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa
     4081 tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg
     4141 aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata
     4201 taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac
     4261 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc
     4321 caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca
     4381 gagacagatc cattcgatta gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa
     4441 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     4501 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     4561 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt ttggttaatt
     4621 taattaaaaa agagacgata tatcgtctcc atctacaaga gtagaaatta cggtgtttcg
     4681 tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat
     4741 atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt
     4801 tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttccaattat
     4861 ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc
     4921 cgctagcggc atgctgggga tgcggtgggc tctatgggtc gagtttactc cctatcagtg
     4981 atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgatgt cgagtttact
     5041 ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat
     5101 gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttatccc tatcagtgat
     5161 agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc gaggtaggcg
     5221 tgtacggtgg gaggcctata taagcagagc tcgtttaacc ggtgccacca tggccccaaa
     5281 gaagaagcgg aaggtcggta tccacggagt cccagcagcc acacagttcg agggctttac
     5341 caacctgtat caggtgagca agacactgcg gtttgagctg atcccacagg gcaagaccct
     5401 gaagcacatc caggagcagg gcttcatcga ggaggacaag gcccgcaatg atcactacaa
     5461 ggagctgaag cccatcatcg atcggatcta caagacctat gccgaccagt gcctgcagct
     5521 ggtgcagctg gattgggaga acctgagcgc cgccatcgac tcctatagaa aggagaaaac
     5581 cgaggagaca aggaacgccc tgatcgagga gcaggccaca tatcgcaatg ccatccacga
     5641 ctacttcatc ggccggacag acaacctgac cgatgccatc aataagagac acgccgagat
     5701 ctacaagggc ctgttcaagg ccgagctgtt taatggcaag gtgctgaagc agctgggcac
     5761 cgtgaccaca accgagcacg agaacgccct gctgcggagc ttcgacaagt ttacaaccta
     5821 cttctccggc ttttatgaga acaggaagaa cgtgttcagc gccgaggata tcagcacagc
     5881 catcccacac cgcatcgtgc aggacaactt ccccaagttt aaggagaatt gtcacatctt
     5941 cacacgcctg atcaccgccg tgcccagcct gcgggagcac tttgagaacg tgaagaaggc
     6001 catcggcatc ttcgtgagca cctccatcga ggaggtgttt tccttccctt tttataacca
     6061 gctgctgaca cagacccaga tcgacctgta taaccagctg ctgggaggaa tctctcggga
     6121 ggcaggcacc gagaagatca agggcctgaa cgaggtgctg aatctggcca tccagaagaa
     6181 tgatgagaca gcccacatca tcgcctccct gccacacaga ttcatccccc tgtttaagca
     6241 gatcctgtcc gataggaaca ccctgtcttt catcctggag gagtttaaga gcgacgagga
     6301 agtgatccag tccttctgca agtacaagac actgctgaga aacgagaacg tgctggagac
     6361 agccgaggcc ctgtttaacg agctgaacag catcgacctg acacacatct tcatcagcca
     6421 caagaagctg gagacaatca gcagcgccct gtgcgaccac tgggatacac tgaggaatgc
     6481 cctgtatgag cggagaatct ccgagctgac aggcaagatc accaagtctg ccaaggagaa
     6541 ggtgcagcgc agcctgaagc acgaggatat caacctgcag gagatcatct ctgccgcagg
     6601 caaggagctg agcgaggcct tcaagcagaa aaccagcgag atcctgtccc acgcacacgc
     6661 cgccctggat cagccactgc ctacaaccct gaagaagcag gaggagaagg agatcctgaa
     6721 gtctcagctg gacagcctgc tgggcctgta ccacctgctg gactggtttg ccgtggatga
     6781 gtccaacgag gtggaccccg agttctctgc ccggctgacc ggcatcaagc tggagatgga
     6841 gccttctctg agcttctaca acaaggccag aaattatgcc accaagaagc cctactccgt
     6901 ggagaagttc aagctgaact ttcagatgcc tacactggcc tctggctggg acgtgaataa
     6961 ggagaagaac aatggcgcca tcctgtttgt gaagaacggc ctgtactatc tgggcatcat
     7021 gccaaagcag aagggcaggt ataaggccct gagcttcgag cccacagaga aaaccagcga
     7081 gggctttgat aagatgtact atgactactt ccctgatgcc gccaagatga tcccaaagtg
     7141 cagcacccag ctgaaggccg tgacagccca ctttcagacc cacacaaccc ccatcctgct
     7201 gtccaacaat ttcatcgagc ctctggagat cacaaaggag atctacgacc tgaacaatcc
     7261 tgagaaggag ccaaagaagt ttcagacagc ctacgccaag aaaaccggcg accagaaggg
     7321 ctacagagag gccctgtgca agtggatcga cttcacaagg gattttctgt ccaagtatac
     7381 caagacaacc tctatcgatc tgtctagcct gcggccatcc tctcagtata aggacctggg
     7441 cgagtactat gccgagctga atcccctgct gtaccacatc agcttccaga gaatcgccga
     7501 gaaggagatc atggatgccg tggagacagg caagctgtac ctgttccaga tctataacaa
     7561 ggactttgcc aagggccacc acggcaagcc taatctgcac acactgtatt ggaccggcct
     7621 gttttctcca gagaacctgg ccaagacaag catcaagctg aatggccagg ccgagctgtt
     7681 ctaccgccct aagtccagga tgaagaggat ggcacaccgg ctgggagaga agatgctgaa
     7741 caagaagctg aaggatcaga aaaccccaat ccccgacacc ctgtaccagg agctgtacga
     7801 ctatgtgaat cacagactgt cccacgacct gtctgatgag gccagggccc tgctgcccaa
     7861 cgtgatcacc aaggaggtgt ctcacgagat catcaaggat aggcgcttta ccagcgacaa
     7921 gttctttttc cacgtgccta tcacactgaa ctatcaggcc gccaattccc catctaagtt
     7981 caaccagagg gtgaatgcct acctgaagga gcaccccgag acacctatca tcggcatcga
     8041 tcggggcgag agaaacctga tctatatcac agtgatcgac tccaccggca agatcctgga
     8101 gcagcggagc ctgaacacca tccagcagtt tgattaccag aagaagctgg acaacaggga
     8161 gaaggagagg gtggcagcaa ggcaggcctg gtctgtggtg ggcacaatca aggatctgaa
     8221 gcagggctat ctgagccagg tcatccacga gatcgtggac ctgatgatcc actaccaggc
     8281 cgtggtggtg ctggagaacc tgaatttcgg ctttaagagc aagaggaccg gcatcgccga
     8341 gaaggccgtg taccagcagt tcgagaagat gctgatcgat aagctgaatt gcctggtgct
     8401 gaaggactat ccagcagaga aagtgggagg cgtgctgaac ccataccagc tgacagacca
     8461 gttcacctcc tttgccaaga tgggcaccca gtctggcttc ctgttttacg tgcctgcccc
     8521 atatacatct aagatcgatc ccctgaccgg cttcgtggac cccttcgtgt ggaaaaccat
     8581 caagaatcac gagagccgca agcacttcct ggagggcttc gactttctgc actacgacgt
     8641 gaaaaccggc gacttcatcc tgcactttaa gatgaacaga aatctgtcct tccagagggg
     8701 cctgcccggc tttatgcctg catgggatat cgtgttcgag aagaacgaga cacagtttga
     8761 cgccaagggc acccctttca tcgccggcaa gagaatcgtg ccagtgatcg agaatcacag
     8821 attcaccggc agataccggg acctgtatcc tgccaacgag ctgatcgccc tgctggagga
     8881 gaagggcatc gtgttcaggg atggctccaa catcctgcca aagctgctgg agaatgacga
     8941 ttctcacgcc atcgacacca tggtggccct gatccgcagc gtgctgcaga tgcggaactc
     9001 caatgccgcc acaggcgagg actatatcaa cagccccgtg cgcgatctga atggcgtgtg
     9061 cttcgactcc cggtttcaga acccagagtg gcccatggac gccgatgcca atggcgccta
     9121 ccacatcgcc ctgaagggcc agctgctgct gaatcacctg aaggagagca aggatctgaa
     9181 gctgcagaac ggcatctcca atcaggactg gctggcctac atccaggagc tgcgcaacaa
     9241 aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagtacc catacgatgt
     9301 tccagattac gctggtaccg gaagcggagg cagtggagag ggcagaggaa gtctgctaac
     9361 atgcggtgac gtcgaggaga atcctggccc atctagaatg gtgagcgagc tgattaagga
     9421 gaacatgcac atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac
     9481 atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca aggcggtcga
     9541 gggcggccct ctccccttcg ccttcgacat cctggctacc agcttcatgt acggcagcaa
     9601 aaccttcatc aaccacaccc agggcatccc cgacttcttt aagcagtcct tccccgaggg
     9661 cttcacatgg gagagagtca ccacatacga agatgggggc gtgctgaccg ctacccagga
     9721 caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttccc
     9781 atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgagacact
     9841 gtaccccgct gacggcggcc tggaaggcag agccgacatg gccctgaagc tcgtgggcgg
     9901 gggccacctg atctgcaacc ttaagaccac atacagatcc aagaaacccg ctaagaacct
     9961 caagatgccc ggcgtctact atgtggacag gagactggaa agaatcaagg aggccgacaa
    10021 agagacatac gtcgagcagc acgaggtggc tgtggccaga tactgcgacc tccctagcaa
    10081 actggggcac aaacttaatt cctagtgatg gtaccgggca gagcgcacat cgcccacagt
    10141 ccccgagaag ttggggggag gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg
    10201 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
    10261 accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag
    10321 aacacagtct agagccacca tgtctaggct ggacaagagc aaagtcataa acggagctct
    10381 ggaattactc aatggtgtcg gtatcgaagg cctgacgaca aggaaactcg ctcaaaagct
    10441 gggagttgag cagcctaccc tgtactggca cgtgaagaac aagcgggccc tgctcgatgc
    10501 cctgccaatc gagatgctgg acaggcatca tacccacttc tgccccctgg aaggcgagtc
    10561 atggcaagac tttctgcgga acaacgccaa gtcataccgc tgtgctctcc tctcacatcg
    10621 cgacggggct aaagtgcatc tcggcacccg cccaacagag aaacagtacg aaaccctgga
    10681 aaatcagctc gcgttcctgt gtcagcaagg cttctccctg gagaacgcac tgtacgctct
    10741 gtccgccgtg ggccacttta cactgggctg cgtattggag gaacaggagc atcaagtagc
    10801 aaaagaggaa agagagacac ctaccaccga ttctatgccc ccacttctga gacaagcaat
    10861 tgagctgttc gaccggcagg gagccgaacc tgccttcctt ttcggcctgg aactaatcat
    10921 atgtggcctg gagaaacagc taaagtgcga aagcggcggg ccgaccgacg cccttgacga
    10981 ttttgactta gacatgctcc cagccgatgc ccttgacgat tttgaccttg acatgctccc
    11041 cgggggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa
    11101 tcctggaccg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
    11161 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
    11221 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
    11281 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
    11341 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
    11401 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
    11461 caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg
    11521 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
    11581 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
    11641 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
    11701 tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac
    11761 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
    11821 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
    11881 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
    11941 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
    12001 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
    12061 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
    12121 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
    12181 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
    12241 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
    12301 cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac
    12361 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg
    12421 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
    12481 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
    12541 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
    12601 tggaaaatct ctagcagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
    12661 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
    12721 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
    12781 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
    12841 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
    12901 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
    12961 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
    13021 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
    13081 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
    13141 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
    13201 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
    13261 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
    13321 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
    13381 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
    13441 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
    13501 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
    13561 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
    13621 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
    13681 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg
    13741 acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa
    13801 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg
    13861 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg
    13921 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg
    13981 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg
    14041 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc
    14101 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg
    14161 ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa
    14221 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
    14281 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
    14341 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
    14401 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
    14461 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
    14521 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
    14581 aactcacatt aattgcgttg cgctcactgc
//
