LOCUS       sequence-299325-        6850 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of AcrIF9 in E. coli, tagged with an HRV3C-cleavage 
            fusion to Maltose Binding Protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6850)
  AUTHORS   Hirschi M, Lu WT, Santiago-Frangos A, Wilkinson R, Golden SM, 
            Davidson AR, Lander GC, Wiedenheft B
  TITLE     AcrIF9 tethers non-sequence specific dsDNA to the CRISPR RNA-guided 
            surveillance complex.
  JOURNAL   Nat Commun. 2020 Jun 1;11(1):2730. doi: 10.1038/s41467-020-16512-1.
  PUBMED    32483187
REFERENCE   2  (bases 1 to 6850)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2020-06-1"; volume: "11(1):2730. doi"; pages: " 
            10.1038/s41467-020-16512-1"
COMMENT     Sequence Label: pMBP-HRV3C-AcrIF9
FEATURES             Location/Qualifiers
     source          1..6850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(167..172,173..188,189..195)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    203..219
                     /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)."
     primer_bind     208..230
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             289..1389
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1363..1386
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1438..1449
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1462..1485
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1702..1719)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1702..1718)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1711..1733)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1897..1914)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2067..2153
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2245..2272
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2291..2382
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2383..2451,2452..3243)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2601..2620)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3285..3798)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3480..3501)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3692..3711
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      3909..4497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4398..4417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4651..4668
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4683..4825
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4751..4770)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4911..4933
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4927..5118)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        5614..5691
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             5692..6774
                     /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     complement(5711..5730)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt
       61 cgtaaatcac tgcataattc gtgtcgctca aggcgcactc ccgttctgga taatgttttt
      121 tgcgccgaca tcataacggt tctggcaaat attctgaaat gagctgttga caattaatca
      181 tcggctcgta taatgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagccag
      241 tccgtttagg tgttttcacg agcacttcac caacaaggac catagcatat gaaaatcgaa
      301 gaaggtaaac tggtaatctg gattaacggc gataaaggct ataacggtct cgctgaagtc
      361 ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc ggataaactg
      421 gaagagaaat tcccacaggt tgcggcaact ggcgatggcc ctgacattat cttctgggca
      481 cacgaccgct ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa
      541 gcgttccagg acaagctgta tccgtttacc tgggatgccg tacgttacaa cggcaagctg
      601 attgcttacc cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga tctgctgccg
      661 aacccgccaa aaacctggga agagatcccg gcgctggata aagaactgaa agcgaaaggt
      721 aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca cctggccgct gattgctgct
      781 gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg
      841 gataacgctg gcgcgaaagc gggtctgacc ttcctggttg acctgattaa aaacaaacac
      901 atgaatgcag acaccgatta ctccatcgca gaagctgcct ttaataaagg cgaaacagcg
      961 atgaccatca acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt gaattatggt
     1021 gtaacggtac tgccgacctt caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc
     1081 gcaggtatta acgccgccag tccgaacaaa gagctggcaa aagagttcct cgaaaactat
     1141 ctgctgactg atgaaggtct ggaagcggtt aataaagaca aaccgctggg tgccgtagcg
     1201 ctgaagtctt acgaggaaga gttggtgaaa gatccgcgga ttgccgccac tatggaaaac
     1261 gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg
     1321 cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc cctgaaagac
     1381 gcgcagacta attcgagctc gaacaacaac aacaataaca ataacaacaa cctcgggatc
     1441 gagggaagga tttcagaatt ccttgaagtt cttttccaag gtcctatgaa aagcacatac
     1501 atcatcaaag aagttcaaaa tatcaattct gaccgtgaag gcgtaaaggt agaaacaaca
     1561 tcattaacta gcgctaaacg tatcgcgtca aaaaaccaat tcttccacgg cactgtacta
     1621 cgcattgagt cagaaagtgg caactggtta gcatataaag aagatggcaa gcgttggatt
     1681 gaatgtgaat aaaagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     1741 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     1801 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggca
     1861 gcttggctgt tttggcggat gagataagat tttcagcctg atacagatta aatcagaacg
     1921 cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga
     1981 ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca
     2041 tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     2101 cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg
     2161 gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat
     2221 aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc
     2281 tacaaactct tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2341 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     2401 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     2461 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     2521 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg tttcccaatg
     2581 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     2641 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     2701 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     2761 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     2821 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2881 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     2941 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     3001 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     3061 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     3121 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     3181 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     3241 taactgtcag accaagttta ctcatatata ctttagattg atttaccccg gttgataatc
     3301 agaaaagccc caaaaacagg aagattgtat aagcaaatat ttaaattgta aacgttaata
     3361 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     3421 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     3481 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     3541 ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt
     3601 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     3661 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     3721 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     3781 cgccgctaca gggcgcgtaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3841 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3901 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3961 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4021 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4081 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4141 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4201 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4261 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4321 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4381 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4441 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4501 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4561 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4621 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4681 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatatggt
     4741 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     4801 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     4861 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4921 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     4981 atcagcgtgg tcgtgcagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     5041 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     5101 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     5161 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     5221 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     5281 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     5341 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     5401 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     5461 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     5521 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     5581 ccgtggccag gacccaacgc tgcccgaaat tccgacacca tcgaatggtg caaaaccttt
     5641 cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca
     5701 gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg
     5761 gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg
     5821 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
     5881 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
     5941 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
     6001 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
     6061 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
     6121 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
     6181 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
     6241 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
     6301 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
     6361 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
     6421 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
     6481 gttggtgcgg atatctcagt agtgggatac gacgataccg aagacagctc atgttatatc
     6541 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
     6601 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
     6661 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     6721 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     6781 cgcaattaat gtaagttagc tcactcatta ggcacaattc tcatgtttga cagcttatca
     6841 tcgactgcac
//
