LOCUS       sequence-177171-        5983 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid contains the gene for anti-CRISPR protein AcrF1 (gene 35 
            from bacteriophage JBD30), with N-terminal 6his tag and TEV protease
            cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5983)
  AUTHORS   Chowdhury S, Carter J, Rollins MF, Golden SM, Jackson RN, Hoffmann 
            C, Nosaka L, Bondy-Denomy J, Maxwell KL, Davidson AR, Fischer ER, 
            Lander GC, Wiedenheft B
  TITLE     Structure Reveals Mechanisms of Viral Suppressors that Intercept a 
            CRISPR RNA-Guided Surveillance Complex.
  JOURNAL   Cell. 2017 Mar 23;169(1):47-57.e11. doi: 10.1016/j.cell.2017.03.012.
  PUBMED    28340349
REFERENCE   2  (bases 1 to 5983)
  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.cell.2017.03"; journalName: "Cell"; date: "2017-03-23- 
            23"; volume: "169"; issue: "1"; pages: "47-57.e11"
COMMENT     Sequence Label: pAcrF1
FEATURES             Location/Qualifiers
     source          1..5983
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             130..150
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             154..177
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(484..502)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      498..545
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(720..725,726..742,743..748))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(775..793)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        861..965
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(966..1034,1035..1826)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1184..1203)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1997..2585
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2486..2505
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2739..2756
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2771..2910
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2836..2855)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2996..3018
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3012..3203)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4515..5597)
                     /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     5559..5578
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5598..5675)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5881..5900
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcagag aaaacttgta tttccagggc atggattaca aggatgacga cgataagttc
      181 atcaaatacc tcagcaccgc tcacctgaac tatatgaata tcgccgttta cgaaaatggc
      241 agcaaaatca aagcccgcgt tgagaacgtc gtaaacggca aaagcgttgg tgctcgtgat
      301 tttgactcaa cggagcaact ggaatcctgg ttttatggtc tgcctggcag tggcctcggt
      361 cgtattgaaa acgctatgaa tgagatttcc cggcgtgaaa acccctgatg acgaagcttg
      421 cggccgcact cgaggatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
      481 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
      541 ttttgctgaa aggaggaact atatccggat atcccgcaag aggcccggca gtaccggcat
      601 aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt
      661 gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca
      721 ttaaagctta tcgatgataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg
      781 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
      841 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
      901 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      961 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     1021 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     1081 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     1141 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     1201 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     1261 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     1321 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     1381 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     1441 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     1501 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     1561 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     1621 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     1681 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     1741 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     1801 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1861 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1921 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1981 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2041 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2101 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     2161 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2221 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2281 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2341 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2401 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2461 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2521 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2581 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2641 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2701 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2761 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2821 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     2881 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     2941 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3001 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     3061 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     3121 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     3181 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     3241 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     3301 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     3361 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     3421 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     3481 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     3541 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     3601 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     3661 cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg
     3721 gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag
     3781 aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca
     3841 ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt
     3901 atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa
     3961 tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc
     4021 cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg
     4081 gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg
     4141 tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct
     4201 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     4261 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     4321 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     4381 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     4441 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     4501 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4561 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     4621 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     4681 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     4741 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     4801 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     4861 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     4921 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     4981 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     5041 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     5101 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     5161 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     5221 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     5281 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     5341 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     5401 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     5461 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     5521 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     5581 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     5641 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     5701 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     5761 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     5821 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     5881 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     5941 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aat
//
