LOCUS       sequence-217563-        7530 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for S. pyogenes Csn2-MBP-His.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7530)
  AUTHORS   Wright AV, Doudna JA
  TITLE     Protecting genome integrity during CRISPR immune adaptation.
  JOURNAL   Nat Struct Mol Biol. 2016 Oct;23(10):876-883. doi: 
            10.1038/nsmb.3289. Epub 2016 Sep 5.
  PUBMED    27595346
REFERENCE   2  (bases 1 to 7530)
  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: "10.1038/nsmb.3289";
            journalName: "Nat Struct Mol Biol"; date: "2016-10"; volume: "23"; 
            issue: "10"; pages: "876-883"
COMMENT     Sequence Label: pAW-SpyCsn2
FEATURES             Location/Qualifiers
     source          1..7530
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     108..127
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     211..230
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        211..229
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    230..254
                     /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             270..292
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             966..986
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             999..2099
                     /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
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2073..2096
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2172..2189
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2242..2260)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2256..2303
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2478..2483,2484..2500,2501..2506))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2533..2551)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2619..2723
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2724..2792,2793..3584)
                     /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(2942..2961)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3755..4343
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4244..4263
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4497..4514
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4529..4668
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4594..4613)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4754..4776
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4770..4961)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     7316..7335
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7355..7432)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
       61 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
      121 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
      181 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
      241 cggataacaa ttcccctcta gagagctctt ttgtttaact ttaagaagga gatatacata
      301 tgaatcttaa tttttcctta ctagatgaac cgattccatt aagaggcggt acaattcttg
      361 tgctcgaaga tgtctgtgta ttttcaaaaa tagtgcaata ttgttaccaa tatgaggaag
      421 attctgaact taaatttttt gatcacaaga tgaaaacaat caaagaatca gaaatcatgc
      481 ttgtaacaga tattttagga tttgatgtta actcctcaac cattttaaaa ttgattcatg
      541 cagatttaga atctcaattt aatgagaaac ccgaagtgaa atcgatgatt gacaaattgg
      601 ttgctacgat tacagaactg attgtctttg aatgcttaga aaatgaatta gatttagagt
      661 atgatgaaat cacaatcctg gaattgatta agtccttagg agtaaaagta gaaacgcaaa
      721 gtgatactat ttttgaaaaa tgtctagaga tacttcaaat tttcaaatat ctcactaaga
      781 aaaagttgct tatttttgtc aatagcggag cttttctaac aaaggatgaa gtggctagtt
      841 tacaagagta tatatcattg acaaatttaa cagttctctt tttagaacca cgtgaactat
      901 atgattttcc gcagtatatt ttagatgaag attatttctt aataactaaa aatatggtag
      961 gttctgaaaa cctgtacttc caatccggat ccggcggcat gaaaatcgaa gaaggtaaac
     1021 tggtaatctg gattaacggc gataaaggct ataacggtct cgctgaagtc ggtaagaaat
     1081 tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat
     1141 tcccacaggt tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct
     1201 ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg
     1261 acaagctgta tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc
     1321 cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga tctgctgccg aacccgccaa
     1381 aaacctggga agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgcgc
     1441 tgatgttcaa cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt
     1501 atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg
     1561 gcgcgaaagc gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag
     1621 acaccgatta ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca
     1681 acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac
     1741 tgccgacctt caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta
     1801 acgccgccag tccgaacaaa gagctggcaa aagagttcct cgaaaactat ctgctgactg
     1861 atgaaggtct ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt
     1921 acgaggaaga gttggcgaaa gatccacgta ttgccgccac tatggaaaac gcccagaaag
     1981 gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg
     2041 tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc cctgaaagac gcgcagacta
     2101 attcgagctc gaacaacaac aacaataaca ataacaacaa cctcgggatc gagggttctt
     2161 ctaaatcttc tcaccatcac catcaccatt gagcggccgc tgctaacaaa gcccgaaagg
     2221 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     2281 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat cccgcaagag
     2341 gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag
     2401 gatgacgatg agtgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa
     2461 ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt
     2521 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     2581 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2641 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2701 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2761 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2821 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2881 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2941 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     3001 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3061 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3121 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3181 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3241 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     3301 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3361 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3421 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3481 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3541 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3601 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3661 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3721 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3781 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3841 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3901 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3961 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4021 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4081 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4141 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4201 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4261 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4321 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4381 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4441 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4501 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4561 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4621 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     4681 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4741 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4801 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     4861 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     4921 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     4981 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     5041 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     5101 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     5161 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     5221 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     5281 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     5341 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     5401 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc
     5461 gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca
     5521 ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg
     5581 aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg
     5641 cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc
     5701 tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg
     5761 taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca
     5821 gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga
     5881 aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca
     5941 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     6001 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     6061 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     6121 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     6181 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     6241 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaact
     6301 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6361 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     6421 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     6481 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     6541 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     6601 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     6661 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     6721 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     6781 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     6841 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     6901 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     6961 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     7021 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     7081 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     7141 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     7201 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     7261 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     7321 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     7381 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     7441 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     7501 ttgagcaccg ccgccgcaag gaatggtgca
//
