LOCUS       sequence-238530-        8817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AcrVA4 in human cells, contained in a lentiviral vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8817)
  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 8817)
  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-AcrVA4-lenti
FEATURES             Location/Qualifiers
     source          1..8817
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        137..516
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..716
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     671..691
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             734..914
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    961..1086
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1579..1812
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1997..2041
                     /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    2339..2456
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2531..3708
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2762..3699
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3656..3676
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3726..3735
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3732..4757
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MAKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             4767..4823
                     /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    5544..6132
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5597..5617)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6015..6026)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6204..6437
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6463..6480)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6469..6693
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6799..6816)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6970..7558)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7050..7069)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7729..8520,8521..8589))
                     /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     8352..8371
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8590..8694)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8769..8788)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
       61 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
      121 gtacgggcca gatattgaca ttgattattg actagttatt aatagtaatc aattacgggg
      181 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      241 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      301 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      361 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      421 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      481 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
      541 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      601 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      661 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg
      721 ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
      781 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
      841 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
      901 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
      961 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
     1021 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
     1081 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
     1141 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
     1201 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
     1261 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
     1321 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     1381 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag
     1441 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
     1501 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
     1561 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
     1621 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
     1681 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
     1741 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
     1801 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
     1861 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
     1921 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
     1981 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
     2041 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
     2101 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
     2161 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag
     2221 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt
     2281 gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc atccacaatt
     2341 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
     2401 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
     2461 tttattacag ggacagcaga gatccagttt ggttaattaa gctagctagg tcttgaaagg
     2521 agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
     2581 gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc gcggggtaaa
     2641 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
     2701 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
     2761 ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt
     2821 gccttgaatt acttccactg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     2881 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     2941 gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     3001 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     3061 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
     3121 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
     3181 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     3241 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     3301 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     3361 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     3421 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     3481 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt
     3541 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     3601 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     3661 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaac cggttctaga
     3721 gcgctgccac catggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     3781 tcgaaaagtt cgacagcgtg tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     3841 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     3901 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
     3961 aagtgcttga cattggggag ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     4021 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
     4081 cggaggcaat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
     4141 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg
     4201 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     4261 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     4321 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     4381 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     4441 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     4501 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     4561 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     4621 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     4681 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     4741 gtccgagggc aaaggaagga tccggcgcaa caaacttctc tctgctgaaa caagccggag
     4801 atgtcgaaga gaatcctgga ccgatgtatg agatcaagct gaacgataca ttgatacacc
     4861 aaactgatga cagagtgaat gccttcgtgg cctatcgcta tcttctgcgg cgcggcgatt
     4921 tgcctaaatg tgaaaacata gcgcggatgt actatgacgg taaagtcatc aaaacagacg
     4981 tgattgacca tgatagtgtt cactccgacg agcaagctaa agtatctaat aacgacataa
     5041 tcaaaatggc catatctgag cttggtgtta ataatttcaa aagtcttata aagaaacaag
     5101 gttacccatt cagcaatggc cacatcaact cttggttcac tgacgacccg gtgaagtcca
     5161 aaactatgca taacgacgag atgtaccttg ttgttcaggc acttataagg gcttgcataa
     5221 tcaaggaaat tgacctgtat actgaacaac tgtataacat tatcaagagt ctgccctatg
     5281 acaaacgacc caatgtagta tactcagacc aacctctgga ccccaacaat ctggacctgt
     5341 ccgaacccga gctttgggct gaacaggtag gcgagtgcat gcggtatgct cataatgacc
     5401 agccttgttt ctatataggt agcacaaaga gagaacttag agtcaactac attgtccccg
     5461 tcatcggcgt aagggatgaa atagagcggg ttatgactct ggaagaggta cgaaatcttc
     5521 ataagtaaac gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga
     5581 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     5641 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     5701 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     5761 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     5821 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     5881 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     5941 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     6001 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     6061 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     6121 ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca
     6181 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct
     6241 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     6301 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     6361 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     6421 gtggaaaatc tctagcaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     6481 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     6541 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     6601 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     6661 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagccatt
     6721 aatgaaaaga accagccatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     6781 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     6841 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     6901 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     6961 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     7021 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     7081 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     7141 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     7201 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     7261 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     7321 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     7381 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     7441 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     7501 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     7561 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     7621 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     7681 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     7741 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     7801 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     7861 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     7921 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     7981 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     8041 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     8101 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     8161 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     8221 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     8281 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     8341 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     8401 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     8461 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     8521 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     8581 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     8641 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     8701 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
     8761 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgct
//
