LOCUS       sequence-218729-        8376 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses AcrIIA4 in human cells, contained in a lentiviral vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8376)
  AUTHORS   Watters KE, Fellmann C, Bai HB, Ren SM, Doudna JA
  TITLE     Systematic discovery of natural CRISPR-Cas12a inhibitors.
  JOURNAL   Science. 2018 Oct 12;362(6411):236-239. doi: 
            10.1126/science.aau5138. Epub 2018 Sep 6.
  PUBMED    30190307
REFERENCE   2  (bases 1 to 8376)
  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"; doi: 
            "10.1126/science.aau5138"; journalName: "Science"; date: 
            "2018-10-12- 12"; volume: "362"; issue: "6411"; pages: "236-239"
COMMENT     Sequence Label: pCF525-EF1a-Hygro-P2A-AcrIIA4-lenti
FEATURES             Location/Qualifiers
     source          1..8376
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        232..611
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        613..811
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     766..786
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             829..1009
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1056..1181
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1674..1907
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2092..2136
                     /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    2434..2551
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2626..3803
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2857..3794
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3751..3771
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3821..3830
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3827..4852
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MAKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             4862..4918
                     /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"
     misc_feature    5198..5786
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5251..5271)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5669..5680)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5858..6091
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6117..6134)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6123..6347
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6453..6470)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6624..7212)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6704..6723)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7383..8174,8175..8243))
                     /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     8006..8025
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8244..8348)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat tgacattgat
      241 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      301 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      361 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      421 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      481 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      541 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      601 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      661 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      721 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      781 ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg
      841 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      901 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      961 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
     1021 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
     1081 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
     1141 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
     1201 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
     1261 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
     1321 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
     1381 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1441 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1501 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1561 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1621 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1681 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1741 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1801 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1861 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1921 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1981 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     2041 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     2101 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     2161 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     2221 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     2281 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     2341 ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact gcgtgcgcca
     2401 attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     2461 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     2521 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     2581 agtttggtta attaagctag ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt
     2641 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
     2701 tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
     2761 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
     2821 gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc
     2881 gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc cactggctgc
     2941 agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt
     3001 gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc
     3061 gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag
     3121 ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa
     3181 atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg
     3241 cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa
     3301 tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt
     3361 gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa
     3421 gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag
     3481 agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt
     3541 catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt
     3601 ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg
     3661 agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg
     3721 ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt
     3781 ttcttccatt tcaggtgtcg tgaaccggtt ctagagcgct gccaccatgg ccaaaaagcc
     3841 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtgtccga
     3901 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     3961 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     4021 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggagttcag
     4081 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     4141 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gcaatggatg cgatcgctgc
     4201 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     4261 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     4321 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     4381 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     4441 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     4501 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     4561 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     4621 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     4681 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     4741 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     4801 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aaggatccgg
     4861 cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgat
     4921 gaatattaat gacttaatta gagaaatcaa aaacaaagat tacacagtga aattgagtgg
     4981 tacggatagc aatagtatca cacagctaat tattcgcgtt aataatgatg gcaacgagta
     5041 tgtaatttct gaaagtgaaa atgaatcaat cgttgaaaaa ttcatctctg cattcaaaaa
     5101 cggttggaat caagaatacg aggatgaaga agaattttat aatgacatgc aaacaatcac
     5161 cttaaaaagt gagttgaact aaacgcgtta agtcgacaat caacctctgg attacaaaat
     5221 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     5281 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     5341 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     5401 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     5461 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     5521 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     5581 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     5641 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     5701 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     5761 gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct
     5821 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     5881 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     5941 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     6001 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     6061 cccttttagt cagtgtggaa aatctctagc agggcccgtt taaacccgct gatcagcctc
     6121 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     6181 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     6241 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     6301 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     6361 aagaaccagc cattaatgaa aagaaccagc cattaatgaa tcggccaacg cgcggggaga
     6421 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     6481 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     6541 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     6601 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6661 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6721 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6781 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6841 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6901 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6961 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     7021 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     7081 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     7141 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     7201 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     7261 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     7321 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     7381 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     7441 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     7501 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     7561 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     7621 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     7681 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7741 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7801 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7861 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7921 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7981 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     8041 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     8101 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     8161 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     8221 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     8281 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     8341 gttccgcgca catttccccg aaaagtgcca cctgac
//
