LOCUS       sequence-225062-        5550 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a 6X-His tagged type II-C anti-CRISPR protein from H. 
            parainfluenzae in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5550)
  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 5550)
  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: pEJS663-pMCSG7-AcrIIC4Hpa
FEATURES             Location/Qualifiers
     source          1..5550
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     38..57
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     145..164
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        145..163
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    164..188
                     /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)."
     RBS             203..225
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             236..253
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             278..298
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             631..648
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(701..719)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      715..762
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(886..905)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1072..1175
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1176..1244,1245..2036)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1394..1413)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2207..2795
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2696..2715
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2949..2966
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2981..3120
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3046..3065)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3206..3228
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3222..3413)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4222..5304)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5266..5285
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5305..5382)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
       61 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
      121 agatcgatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
      181 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac atatgcacca
      241 tcatcatcat cattcttctg gtgtagatct gggtaccgag aacctgtact tccaatccat
      301 gaagatcacc agcagcaact tcgcgaccat tgcgaccagc gagaactttg cgaagctgag
      361 cgtgctgccg aaaaaccacc gtgagccgat caagggtctg ttcaaaagcg cggttgaaca
      421 gtttagcagc gcgcgtgact tctttaagaa cgagaactac agcaaagagc tggcggaaaa
      481 gttcaacaaa gaagcggtga acgaggcggt tgaaaagctg caaaaagcga tcgatctggc
      541 ggaaaaacag ggcattcaat tttaaaacat tggaagtgga taacggatcc gaattcgagc
      601 tccgtcgaca agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct
      661 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
      721 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
      781 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      841 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
      901 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
      961 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     1021 atggttcaca ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     1081 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     1141 tgataaatgc ttcaataatt tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     1201 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     1261 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     1321 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     1381 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     1441 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     1501 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     1561 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1621 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1681 aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
     1741 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     1801 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     1861 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     1921 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     1981 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     2041 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     2101 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     2161 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2221 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2281 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2341 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2401 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2461 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2521 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2581 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2641 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2701 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2761 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2821 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcttgcgtta tcccctgatt
     2881 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     2941 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     3001 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     3061 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3121 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3181 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3241 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     3301 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     3361 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     3421 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     3481 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     3541 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     3601 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     3661 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     3721 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gttgcttcac
     3781 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     3841 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     3901 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     3961 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     4021 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     4081 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     4141 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     4201 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4261 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4321 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4381 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4441 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     4501 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     4561 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     4621 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     4681 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     4741 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     4801 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     4861 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     4921 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     4981 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5041 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5101 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5161 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5221 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5281 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     5341 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     5401 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     5461 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     5521 ctgccaccat acccacgccg aaacaagcgc
//
