LOCUS       sequence-225602-        8376 bp    DNA     circular SYN 15-DEC-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 Dec 15, 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"
     enhancer        106..485
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        487..685
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     640..660
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             703..883
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    930..1055
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1548..1781
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1966..2010
                     /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    2308..2425
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2500..3677
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2731..3668
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3625..3645
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3695..3704
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3701..4726
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MAKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             4736..4792
                     /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    5072..5660
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5125..5145)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5543..5554)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5732..5965
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5991..6008)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5997..6221
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6327..6344)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6498..7086)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6578..6597)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7257..8048,8049..8117))
                     /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     7880..7899
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8118..8222)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8297..8316)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
       61 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atattgacat tgattattga
      121 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      181 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      241 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      301 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      361 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      421 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      481 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      541 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      601 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      661 tacggtggga ggtctatata agcagcgcgt tttgcctgta ctgggtctct ctggttagac
      721 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
      781 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
      841 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg
      901 acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag
      961 cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg
     1021 gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat
     1081 cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata
     1141 tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat
     1201 cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag
     1261 aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga
     1321 taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca
     1381 ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg
     1441 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
     1501 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
     1561 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
     1621 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
     1681 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
     1741 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
     1801 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg
     1861 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
     1921 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
     1981 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
     2041 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
     2101 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
     2161 cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag
     2221 agagacagag acagatccat tcgattagtg aacggatcgg cactgcgtgc gccaattctg
     2281 cagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     2341 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     2401 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg
     2461 gttaattaag ctagctaggt cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg
     2521 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgatcc
     2581 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
     2641 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
     2701 tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct
     2761 ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccactgg ctgcagtacg
     2821 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt
     2881 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
     2941 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt
     3001 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg
     3061 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
     3121 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac
     3181 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg
     3241 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
     3301 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg
     3361 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg
     3421 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
     3481 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg
     3541 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt
     3601 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
     3661 catttcaggt gtcgtgaacc ggttctagag cgctgccacc atggccaaaa agcctgaact
     3721 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtgt ccgacctgat
     3781 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     3841 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca
     3901 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggagt tcagcgagag
     3961 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     4021 cgaactgccc gctgttctgc agccggtcgc ggaggcaatg gatgcgatcg ctgcggccga
     4081 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     4141 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     4201 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     4261 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     4321 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     4381 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     4441 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     4501 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     4561 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     4621 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     4681 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaaggat ccggcgcaac
     4741 aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac cgatgaatat
     4801 taatgactta attagagaaa tcaaaaacaa agattacaca gtgaaattga gtggtacgga
     4861 tagcaatagt atcacacagc taattattcg cgttaataat gatggcaacg agtatgtaat
     4921 ttctgaaagt gaaaatgaat caatcgttga aaaattcatc tctgcattca aaaacggttg
     4981 gaatcaagaa tacgaggatg aagaagaatt ttataatgac atgcaaacaa tcaccttaaa
     5041 aagtgagttg aactaaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga
     5101 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     5161 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     5221 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     5281 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     5341 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     5401 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     5461 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     5521 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     5581 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     5641 cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat
     5701 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     5761 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     5821 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     5881 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     5941 tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc cgctgatcag cctcgactgt
     6001 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     6061 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     6121 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     6181 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     6241 cagccattaa tgaaaagaac cagccattaa tgaatcggcc aacgcgcggg gagaggcggt
     6301 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     6361 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     6421 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     6481 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     6541 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     6601 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     6661 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6721 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6781 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6841 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6901 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     6961 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     7021 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     7081 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     7141 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     7201 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     7261 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     7321 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     7381 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     7441 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     7501 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     7561 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     7621 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     7681 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     7741 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7801 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7861 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7921 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7981 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     8041 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     8101 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     8161 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     8221 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     8281 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     8341 ctgcttgtgt gttggaggtc gctgagtagt gcgcga
//
