LOCUS       sequence-224369-        8391 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AcrVA5 in human cells, contained in a lentiviral vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8391)
  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 8391)
  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-AcrVA5-lenti
FEATURES             Location/Qualifiers
     source          1..8391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        79..458
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        460..658
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     613..633
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             676..856
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    903..1028
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1521..1754
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1939..1983
                     /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    2281..2398
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2473..3650
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2704..3641
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3598..3618
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3668..3677
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3674..4699
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MAKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             4709..4765
                     /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    5060..5648
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5113..5133)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5531..5542)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5720..5953
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5979..5996)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5985..6209
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6315..6332)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6486..7074)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6566..6585)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7245..8036,8037..8105))
                     /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     7868..7887
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8106..8210)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8285..8304)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
       61 atgtacgggc cagatattga cattgattat tgactagtta ttaatagtaa tcaattacgg
      121 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
      181 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
      241 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
      301 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
      361 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
      421 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
      481 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
      541 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
      601 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg
      661 cgttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
      721 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
      781 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
      841 tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg
      901 agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc
      961 gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg
     1021 agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc
     1081 cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac
     1141 gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac
     1201 agctacaacc atcccttcag acaggatcag aagaacttag atcattatat aatacagtag
     1261 caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca
     1321 agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc
     1381 agacctggag gaggagatat gagggacaat tggagaagtg aattatataa atataaagta
     1441 gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga
     1501 gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc
     1561 actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata
     1621 gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc
     1681 acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag
     1741 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg
     1801 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg
     1861 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa
     1921 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt
     1981 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata
     2041 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt
     2101 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac
     2161 aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta
     2221 gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa atggcagtat tcatccacaa
     2281 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
     2341 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
     2401 ggtttattac agggacagca gagatccagt ttggttaatt aagctagcta ggtcttgaaa
     2461 ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
     2521 gagaagttgg ggggaggggt cggcaattga tccggtgcct agagaaggtg gcgcggggta
     2581 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
     2641 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
     2701 caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc
     2761 gtgccttgaa ttacttccac tggctgcagt acgtgattct tgatcccgag cttcgggttg
     2821 gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag
     2881 ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct
     2941 gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc
     3001 tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg
     3061 tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc
     3121 ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg
     3181 ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc
     3241 ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct
     3301 caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa
     3361 gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca
     3421 ggcacctcga ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt
     3481 tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc
     3541 acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca
     3601 agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga accggttcta
     3661 gagcgctgcc accatggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     3721 gatcgaaaag ttcgacagcg tgtccgacct gatgcagctc tcggagggcg aagaatctcg
     3781 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
     3841 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
     3901 ggaagtgctt gacattgggg agttcagcga gagcctgacc tattgcatct cccgccgtgc
     3961 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt
     4021 cgcggaggca atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
     4081 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
     4141 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
     4201 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
     4261 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
     4321 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
     4381 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
     4441 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
     4501 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
     4561 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
     4621 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
     4681 tcgtccgagg gcaaaggaag gatccggcgc aacaaacttc tctctgctga aacaagccgg
     4741 agatgtcgaa gagaatcctg gaccgatgaa gatcgagctt tccggggggt atatttgtta
     4801 cagcattgag gaagacgaag ttactataga catggtagag gtcacgacta aacgccaggg
     4861 gataggctct cagcttatag atatggtaaa ggatgtggct agagaagtag gtctcccgat
     4921 aggtctttat gcgtatcctc aagatgacag tattagtcag gaggatctga tcgagtttta
     4981 cttcagtaat gattttgagt atgacccaga cgatgtcgat ggacgcctta tgcgctggtc
     5041 ttaaacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     5101 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     5161 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     5221 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     5281 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     5341 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     5401 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     5461 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     5521 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     5581 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     5641 ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     5701 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
     5761 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     5821 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     5881 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     5941 aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     6001 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     6061 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     6121 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     6181 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gccattaatg
     6241 aaaagaacca gccattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     6301 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     6361 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     6421 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     6481 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     6541 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     6601 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6661 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6721 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6781 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6841 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6901 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6961 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     7021 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     7081 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     7141 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     7201 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     7261 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     7321 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     7381 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     7441 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     7501 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     7561 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     7621 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     7681 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7741 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7801 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7861 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7921 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7981 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     8041 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     8101 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     8161 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     8221 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     8281 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     8341 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa g
//
