LOCUS       sequence-177169-        6016 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid contains the gene for anti-CRISPR protein AcrF2 (gene 30 
            from bacteriophage D3112), 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 6016)
  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 6016)
  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: pAcrF2
FEATURES             Location/Qualifiers
     source          1..6016
                     /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"
     primer_bind     complement(517..535)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      531..578
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(753..758,759..775,776..781))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(808..826)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        894..998
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(999..1067,1068..1859)
                     /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(1217..1236)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2030..2618
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2519..2538
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2772..2789
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2804..2943
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2869..2888)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3029..3051
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3045..3236)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4548..5630)
                     /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     5592..5611
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5631..5708)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5914..5933
                     /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 atgactaaga ccgcacaaat gatcgctcag
      181 cagcacaaag atactgtcgc agcgtgcgaa gccgccgaag ccatcgctat cgctaaagat
      241 caggtatggg atggcgaggg ctataccaag tacacgttcg acgacaacag cgtcctgatc
      301 cagtcgggca ctactcagta tgcgatggat gccgacgacg cagacagcat caaaggctat
      361 gcggactggc tggacgatga ggctcgctcc gccgaagcgt cggagatcga gcgcctgctt
      421 gaatcggtcg aggaggagtg atgacgaagc ttgcggccgc actcgaggat ccggctgcta
      481 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
      541 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
      601 gatatcccgc aagaggcccg gcagtaccgg cataaccaag cctatgccta cagcatccag
      661 ggtgacggtg ccgaggatga cgatgagcgc attgttagat ttcatacacg gtgcctgact
      721 gcgttagcaa tttaactgtg ataaactacc gcattaaagc ttatcgatga taagctgtca
      781 aacatgagaa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
      841 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
      901 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
      961 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     1021 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     1081 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     1141 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1201 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc
     1261 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1321 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     1381 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1441 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     1501 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     1561 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1621 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     1681 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     1741 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     1801 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     1861 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     1921 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     1981 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2041 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2101 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2161 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2221 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2281 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2341 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2401 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2461 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2521 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2581 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2641 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2701 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2761 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     2821 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     2881 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     2941 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3001 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3061 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     3121 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     3181 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     3241 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     3301 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     3361 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     3421 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     3481 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     3541 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     3601 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     3661 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg
     3721 cccgagatgc gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa
     3781 gggttggttt gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg
     3841 gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg
     3901 caccgcgacg caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca
     3961 acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga
     4021 tcgaagttag gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat
     4081 ctacctgcct ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa
     4141 gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca
     4201 gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     4261 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     4321 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     4381 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     4441 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     4501 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     4561 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4621 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     4681 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     4741 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     4801 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     4861 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     4921 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     4981 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     5041 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     5101 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     5161 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     5221 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     5281 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     5341 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     5401 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     5461 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     5521 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     5581 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     5641 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     5701 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     5761 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     5821 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     5881 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     5941 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     6001 ctcgatcccg cgaaat
//
