LOCUS       sequence-217555-        7730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for His-MBP-S. pyogenes Cas1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7730)
  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 7730)
  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-SpyCas1
FEATURES             Location/Qualifiers
     source          1..7730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     73..92
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        73..91
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    92..116
                     /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             132..154
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             174..191
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             201..1301
                     /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     1275..1298
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1356..1376
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2304..2322)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2318..2365
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2540..2545,2546..2562,2563..2568))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2595..2613)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2681..2785
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2786..2854,2855..3646)
                     /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(3004..3023)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3817..4405
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4306..4325
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4559..4576
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4591..4730
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4656..4675)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4816..4838
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4832..5023)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     7378..7397
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7417..7494)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7700..7719
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
       61 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
      121 tagagagctc ttttgtttaa ctttaagaag gagatataca tatgaaatct tctcaccatc
      181 accatcacca tggttcttct atgaaaatcg aagaaggtaa actggtaatc tggattaacg
      241 gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa gataccggaa
      301 ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag gttgcggcaa
      361 ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc tacgctcaat
      421 ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg tatccgttta
      481 cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct gttgaagcgt
      541 tatcgctgat ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc
      601 cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag
      661 aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa
      721 acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga
      781 ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat tactccatcg
      841 cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg tgggcatggt
      901 ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc ttcaagggtc
      961 aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca
     1021 aagagctggc aaaagagttc ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg
     1081 ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga
     1141 aagatccacg tattgccgcc actatggaaa acgcccagaa aggtgaaatc atgccgaaca
     1201 tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg
     1261 gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac taattcgagc tcgaacaaca
     1321 acaacaataa caataacaac aacctcggga tcgaggaaaa cctgtacttc caaggatcca
     1381 tggctggttg gcgtactgtt gtggtaaata cccactcgaa attatcctat aagaataatc
     1441 atctgatttt taaggatgcc tataaaacgg agctgatcca tttatcagaa attgatattt
     1501 tgttattaga aacgaccgat attgtcttgt ccactatgct ggtaaaacgg ctagtggatg
     1561 agaatgtcct tgtcatattc tgtgatgata aacgattacc aacagctatg ctgatgcctt
     1621 tttatggtcg tcatgattcg agtttacagc ttgggaaaca aatgtcctgg tcagaaacag
     1681 tcaaatcgca ggtttggacg acgattattg ctcaaaagat tttgaatcaa tcttgctatc
     1741 taggagcatg ctcctatttt gaaaaatccc aatctattat ggatttatat catggtttgg
     1801 aaaattttga tccgagtaat cgagaagggc atgcagcgag aatttatttt aatacacttt
     1861 ttgggaacga tttctcaaga gatttggagc atccaatcaa tgcaggtctg gattatggtt
     1921 atactttatt attgagtatg tttgcgcgtg aagtggttgt gtctggatgt atgactcagt
     1981 ttgggcttaa acacgctaat cagtttaatc agttcaattt tgctagcgat attatggaac
     2041 catttaggcc tttagtggat aagattgttt atgaaaatcg aaatcagcct tttcccaaaa
     2101 taaagagaga gttatttact ttgttttcag atacattttc atataatggt aaagagatgt
     2161 atctcacgaa tattattagc gattatacta aaaaagttgt caaagctctg aataatgaag
     2221 ggaaaggagt tcctgaattt aggatatgac tcgagcggcc gctgctaaca aagcccgaaa
     2281 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     2341 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat atcccgcaag
     2401 aggcccggca gtaccggcat aaccaagcct atgcctacag catccagggt gacggtgccg
     2461 aggatgacga tgagtgcatt gttagatttc atacacggtg cctgactgcg ttagcaattt
     2521 aactgtgata aactaccgca ttaaagctta tcgatgataa gctgtcaaac atgagaattc
     2581 ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     2641 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2701 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2761 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2821 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     2881 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     2941 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     3001 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     3061 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     3121 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3181 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3241 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3301 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     3361 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3421 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3481 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     3541 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3601 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3661 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3721 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3781 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3841 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3901 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3961 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4021 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4081 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4141 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4201 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4261 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4321 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4381 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4441 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4501 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4561 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     4621 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4681 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     4741 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     4801 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     4861 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     4921 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     4981 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     5041 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     5101 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     5161 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     5221 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     5281 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     5341 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     5401 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     5461 acgacaggag cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc
     5521 gcgtgcggct gctggagatg gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg
     5581 cattcacagt tctccgcaag aattgattgg ctccaattct tggagtggtg aatccgttag
     5641 cgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa
     5701 cgcggggagg cagacaaggt atagggcggc gcctacaatc catgccaacc cgttccatgt
     5761 gctcgccgag gcggcataaa tcgccgtgac gatcagcggt ccaatgatcg aagttaggct
     5821 ggtaagagcc gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta cctgcctgga
     5881 cagcatggcc tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa tcataatggg
     5941 gaaggccatc cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc
     6001 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     6061 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     6121 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     6181 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     6241 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     6301 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     6361 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     6421 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     6481 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     6541 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     6601 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     6661 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     6721 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     6781 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     6841 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     6901 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     6961 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     7021 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     7081 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     7141 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     7201 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     7261 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     7321 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     7381 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     7441 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     7501 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     7561 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     7621 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     7681 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
//
