LOCUS       sequence-263524-        7994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses type II-A anti-CRISPR protein AcrIIA5 in mammalian cells 
            in a lentiviral vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7994)
  AUTHORS   Garcia B, Lee J, Edraki A, Hidalgo-Reyes Y, Erwood S, Mir A, Trost 
            CN, Seroussi U, Stanley SY, Cohn RD, Claycomb JM, Sontheimer EJ, 
            Maxwell KL, Davidson AR
  TITLE     Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for 
            Genome Editing.
  JOURNAL   Cell Rep. 2019 Nov 12;29(7):1739-1746.e5. doi: 
            10.1016/j.celrep.2019.10.017.
  PUBMED    31722192
REFERENCE   2  (bases 1 to 7994)
  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: 
            "10.1016/j.celrep.2019.10"; journalName: "Cell Rep"; date: 
            "2019-11-12- 12"; volume: "29"; issue: "7"; pages: "1739-1746.e5"
COMMENT     Sequence Label: pEJS1005-Lenti-mammalian c.o.AcrIIA5-FLAG-NLS-...
FEATURES             Location/Qualifiers
     source          1..7994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      8..17
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             440..463
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             470..490
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        514..725
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      735..744
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             741..1769
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             741..1769
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     misc_feature    1783..2371
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1836..1856)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2254..2265)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(2447..2480)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2685..2702)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2691..2915
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2988..3005)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3159..3747)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3239..3258)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3918..4709,4710..4778))
                     /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     4541..4560
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4779..4883)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4917..5143
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5144..5324
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5371..5496
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5988..6221
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6406..6450
                     /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    6717..6834
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        7576..7982
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
ORIGIN
        1 cgaagccgcc accatggctt atggtaaatc tcggtataat tcttatagga aaaggagctt
       61 caatcgctct aataaacaac gccgggagta cgctcaggaa atggacagac tggagaaggc
      121 gtttgagaat cttgatggat ggtacttgtc atcaatgaaa gactcagcct acaaagactt
      181 tgggaagtat gagatccggc tgtctaacca cagcgcagat aacaagtacc atgatctcga
      241 aaatggacga ctcattgtca atatcaaagc ttcaaagctg aacttcgtcg atattataga
      301 gaacaaactg gacaaaatca tagagaaaat cgacaaattg gatctcgaca agtatcgatt
      361 tataaatgca acgaatcttg aacatgacat caaatgttat tacaagggtt tcaaaactaa
      421 aaaggaggtc attggtggtg attataagga tgacgatgac aaaggcagcc cgaagaagaa
      481 aaggaaggtg tgactcgagc ctctagaact atagggcaga gcgcacatcg cccacagtcc
      541 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
      601 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
      661 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
      721 cacaggatcc tcgagccacc atgggtaaaa agcctgaact caccgcgacg tctgtcgaga
      781 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
      841 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
      901 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
      961 cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc
     1021 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
     1081 agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
     1141 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
     1201 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
     1261 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
     1321 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
     1381 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
     1441 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
     1501 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
     1561 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
     1621 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
     1681 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
     1741 ccagcactcg tccgagggca aaggaataaa tctagtgtcg acaatcaacc tctggattac
     1801 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     1861 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     1921 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     1981 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     2041 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     2101 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     2161 gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg
     2221 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     2281 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     2341 agtcggatct ccctttgggc cgcctccccg cctggaattc ggtacctttt taaaagaaaa
     2401 ggggggactg gaagggctaa ttcactccca acgaagacaa gatatcataa cttcgtatag
     2461 catacattat acgaagttat aatttatttg tgaaatttgt gatgctattg ctttatttgt
     2521 aaccatatgt ttatttgtga aatttgtgat gctattgctt tatttgtaac cattgctttt
     2581 tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     2641 agggaaccca ctgcttaagc ctcaataaag cttgcctcga ccagcctcga ctgtgccttc
     2701 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     2761 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     2821 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2881 tagcaggcat gctggggatg cggtgggctc tatggcctgc agctgcatta atgaatcggc
     2941 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3001 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3061 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3121 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3181 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3241 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3301 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3361 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3421 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3481 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3541 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     3601 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3661 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3721 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3781 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3841 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3901 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3961 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4021 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4081 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4141 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4201 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4261 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4321 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4381 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4441 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4501 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4561 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4621 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4681 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4741 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4801 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4861 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcaatgta
     4921 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt
     4981 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc
     5041 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca
     5101 ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga
     5161 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     5221 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     5281 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
     5341 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
     5401 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
     5461 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
     5521 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
     5581 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
     5641 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     5701 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     5761 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     5821 accgcacagc aagcggccct gatcttcaga cctggaggag gagatatgag ggacaattgg
     5881 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
     5941 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
     6001 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
     6061 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
     6121 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
     6181 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
     6241 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa tgaatctctg
     6301 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
     6361 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
     6421 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
     6481 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
     6541 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
     6601 cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag
     6661 agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg gttaactttt
     6721 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     6781 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     6841 tattacaggg acagcagaga tccagtttgg atcgataagc ttgatatcga attcgggagg
     6901 tggtccctgc agttacgcca atgataaccc ccgccagaaa aatcttagta gccttccctt
     6961 tttgttttcc gtgccccaac tcggcggatt gactcggccc cttccggaaa cacccgaatc
     7021 aacttctagt caaattattg ttcacgccgc aatgacccac ccctggcccg cgtctgtgga
     7081 actgacccct ggtgtacagg agagttcgct gctgaaagtg gtcccaaagg ggtactagtt
     7141 tttaagctcc caactccccc tcccccagcg tctggaggat tccacaccct cgcaccgcag
     7201 gggcgaggaa gtgggcggag tccggttttg gcgccagccg ctgaggctgc caagcagaaa
     7261 agccaccgct gaggagactc cggtcactgt cctcgccccg cctccccctt ccctcccctt
     7321 ggggaccacc gggcgccacg ccgcgaacgg taagtgccgc ggtcgtcggc gcctccgccc
     7381 tccccctagg gccccaattc ccagcgggcg cggcgcgcgg ccccgcgcgc agctcccggc
     7441 tccctccccc ttcggatgtg gcttgagctg taggcgcgga gtcctgcagc cccgataaaa
     7501 taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt
     7561 ggcaagctag ctgcagtaac gccattttgc aaggcatgga aaaataccaa accaagaata
     7621 gagaagttca gatcaagggc gggtacatga aaatagctaa cgttgggcca aacaggatat
     7681 ctgcggtgag cagtttcggc cccggcccgg ggccaagaac agatggtcac cgcagtttcg
     7741 gccccggccc gaggccaaga acagatggtc cccagatatg gcccaaccct cagcagtttc
     7801 ttaagaccca tcagatgttt ccaggctccc ccaaggacct gaaatgaccc tgcgccttat
     7861 ttgaattaac caatcagcct gcttctcgct tctgttcgcg cgcttctgct tcccgagctc
     7921 tataaaagag ctcacaaccc ctcactcggc gcgccagtcc tccgacagac tgagtcgccc
     7981 ggatcccatc gatt
//
