LOCUS       sequence-225078-        4415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a FLAG/NLS tagged type II-C anti-CRISPR protein from H. 
            parainfluenzae in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4415)
  AUTHORS   Lee J, Mir A, Edraki A, Garcia B, Amrani N, Lou HE, Gainetdinov I, 
            Pawluk A, Ibraheim R, Gao XD, Liu P, Davidson AR, Maxwell KL, 
            Sontheimer EJ
  TITLE     Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and 
            AcrIIC5 Anti-CRISPR Proteins.
  JOURNAL   MBio. 2018 Dec 4;9(6). pii: mBio.02321-18. doi: 
            10.1128/mBio.02321-18.
  PUBMED    30514786
REFERENCE   2  (bases 1 to 4415)
  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"; journalName: "MBio."; 
            date: "2018-12-4"; volume: "9(6). pii: mBio.02321-18. doi"; pages: "
            10.1128/mBio.02321-18"
COMMENT     Sequence Label: pEJS581-pCSDest2-AcrIIC4Hpa-FLAG-NLS
FEATURES             Location/Qualifiers
     source          1..4415
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        67..1051
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1086..1104
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1086..1103
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1427..1450
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1457..1477
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1518..1652
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1542..1561)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1596..1615
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1765..1785)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1765..1783)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1804..1820)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1804..1820)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1817..1839)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1828..1844
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1852..1858,1859..1876,1877..1882))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1897..1918
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2035..2052)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2206..2794)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2286..2305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2965..3756,3757..3825))
                     /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     3588..3607
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3826..3930)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3956..4411)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4093..4114)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4305..4324
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
       61 acgccagtcg accgccaatt caatatggcg tatatggact catgccaatt caatatggtg
      121 gatctggacc tgtgccaatt caatatggcg tatatggact cgtgccaatt caatatggtg
      181 gatctggacc ccagccaatt caatatggcg gacttggcac catgccaatt caatatggcg
      241 gacctggcac tgtgccaact ggggaggggt ctacttggca cggtgccaag tttgaggagg
      301 ggtcttggcc ctgtgccaag tccgccatat tgaattggca tggtgccaat aatggcggcc
      361 atattggcta tatgccagga tcaatatata ggcaatatcc aatatggccc tatgccaata
      421 tggctattgg ccaggttcaa tactatgtat tggccctatg ccatatagta ttccatatat
      481 gggttttcct attgacgtag atagcccctc ccaatgggcg gtcccatata ccatatatgg
      541 ggcttcctaa taccgcccat agccactccc ccattgacgt caatggtctc tatatatggt
      601 ctttcctatt gacgtcatat gggcggtcct attgacgtat atggcgcctc ccccattgac
      661 gtcaattacg gtaaatggcc cgcctggctc aatgcccatt gacgtcaata ggaccaccca
      721 ccattgacgt caatgggatg gctcattgcc cattcatatc cgttctcacg ccccctattg
      781 acgtcaatga cggtaaatgg cccacttggc agtacatcaa tatctattaa tagtaacttg
      841 gcaagtacat tactattgga agtacgccag ggtacattgg cagtactccc attgacgtca
      901 atggcggtaa atggcccgcg atggctgcca agtacatccc cattgacgtc aatggggagg
      961 ggcaatgacg caaatgggcg ttccattgac gtaaatgggc ggtaggcgtg cctaatggga
     1021 ggtctatata agcaatgctc gtttagggaa ccgccattct gcctggggac gtcggagcaa
     1081 gcttgattta ggtgacacta tagaatacaa gctacttgtt ctttttgcag gatcccatcg
     1141 attcgaagcc gccaccatga agatcaccag cagcaacttc gcgaccattg cgaccagcga
     1201 gaactttgcg aagctgagcg tgctgccgaa aaaccaccgt gagccgatca agggtctgtt
     1261 caaaagcgcg gttgaacagt ttagcagcgc gcgtgacttc tttaagaacg agaactacag
     1321 caaagagctg gcggaaaagt tcaacaaaga agcggtgaac gaggcggttg aaaagctgca
     1381 aaaagcgatc gatctggcgg aaaaacaggg cattcaattt ggcggggatt ataaggatga
     1441 tgatgataag ggctccccta agaagaagag aaaggtgtga ctcgagcctc tagaactata
     1501 gtgagtcgta ttacgtagat ccagacatga taagatacat tgatgagttt ggacaaacca
     1561 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     1621 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     1681 ttcaggttca gggggaggtg tgggaggttt tttaattcgc ggccgcggcg ccaatgcatt
     1741 gggcccggta cccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     1801 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     1861 agccgggagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     1921 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     1981 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     2041 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2101 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2161 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2221 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2281 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2341 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2401 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     2461 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2521 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     2581 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     2641 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     2701 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     2761 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     2821 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     2881 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     2941 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3001 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3061 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     3121 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3181 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     3241 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     3301 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     3361 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     3421 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     3481 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3541 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     3601 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     3661 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     3721 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     3781 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     3841 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     3901 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
     3961 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     4021 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     4081 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     4141 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     4201 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     4261 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     4321 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     4381 gccgcgctta atgcgccgct acagggcgcg tccca
//
