LOCUS       sequence-217571-        7202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for His-MBP-S. pyogenes Cas2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7202)
  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 7202)
  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-SpyCas2
FEATURES             Location/Qualifiers
     source          1..7202
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     45..64
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        45..63
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    64..88
                     /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             104..126
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             146..163
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             173..1273
                     /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     1247..1270
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1328..1348
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1748..1766)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1762..1809
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1984..1989,1990..2006,2007..2012))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2039..2057)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2125..2229
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2230..2298,2299..3090)
                     /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(2448..2467)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3261..3849
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3750..3769
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4003..4020
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4035..4174
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4100..4119)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4260..4282
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4276..4467)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6822..6841
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6861..6938)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7144..7163
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
       61 aggggaattg tgagcggata acaattcccc tctagagagc tcttttgttt aactttaaga
      121 aggagatata catatgaaat cttctcacca tcaccatcac catggttctt ctatgaaaat
      181 cgaagaaggt aaactggtaa tctggattaa cggcgataaa ggctataacg gtctcgctga
      241 agtcggtaag aaattcgaga aagataccgg aattaaagtc accgttgagc atccggataa
      301 actggaagag aaattcccac aggttgcggc aactggcgat ggccctgaca ttatcttctg
      361 ggcacacgac cgctttggtg gctacgctca atctggcctg ttggctgaaa tcaccccgga
      421 caaagcgttc caggacaagc tgtatccgtt tacctgggat gccgtacgtt acaacggcaa
      481 gctgattgct tacccgatcg ctgttgaagc gttatcgctg atttataaca aagatctgct
      541 gccgaacccg ccaaaaacct gggaagagat cccggcgctg gataaagaac tgaaagcgaa
      601 aggtaagagc gcgctgatgt tcaacctgca agaaccgtac ttcacctggc cgctgattgc
      661 tgctgacggg ggttatgcgt tcaagtatga aaacggcaag tacgacatta aagacgtggg
      721 cgtggataac gctggcgcga aagcgggtct gaccttcctg gttgacctga ttaaaaacaa
      781 acacatgaat gcagacaccg attactccat cgcagaagct gcctttaata aaggcgaaac
      841 agcgatgacc atcaacggcc cgtgggcatg gtccaacatc gacaccagca aagtgaatta
      901 tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct
      961 gagcgcaggt attaacgccg ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa
     1021 ctatctgctg actgatgaag gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt
     1081 agcgctgaag tcttacgagg aagagttggc gaaagatcca cgtattgccg ccactatgga
     1141 aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt tctggtatgc
     1201 cgtgcgtact gcggtgatca acgccgccag cggtcgtcag actgtcgatg aagccctgaa
     1261 agacgcgcag actaattcga gctcgaacaa caacaacaat aacaataaca acaacctcgg
     1321 gatcgaggaa aacctgtact tccaaggatc catgagttat agatatatga gaatgatact
     1381 tatgtttgat atgccgacgg acaccgctga ggaacgaaaa gcctatcgaa aatttcggaa
     1441 atttttactt agtgaagggt ttatcatgca tcaattttct atttatagta agttgctgtt
     1501 gaataataca gctaacaatg ccatgattgg tcggctgagg gagcataatc ctaataaagg
     1561 aaatattaca ttactaacgg tcacggaaaa acagtttgca cgaatgattt atttacatgg
     1621 tgaaagaaat aattgtattg caaactccga tgaaagactt gtatttcttg gggaggcttt
     1681 tgatgaatct taactcgagc ggccgctgct aacaaagccc gaaaggaagc tgagttggct
     1741 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1801 ggttttttgc tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg
     1861 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagtg
     1921 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
     1981 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc
     2041 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     2101 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2161 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2221 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2281 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2341 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2401 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2461 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2521 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2581 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2641 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2701 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2761 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2821 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2881 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2941 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3001 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3061 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3121 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3181 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3241 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3301 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3361 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3421 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3481 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3541 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3601 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3661 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3721 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3781 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3841 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3901 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3961 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4021 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4081 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4141 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4201 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4261 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4321 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     4381 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     4441 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     4501 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     4561 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     4621 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4681 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     4741 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     4801 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     4861 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     4921 catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga
     4981 gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg
     5041 caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct
     5101 tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca
     5161 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca
     5221 taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc
     5281 gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac
     5341 gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct
     5401 cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
     5461 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     5521 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     5581 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     5641 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     5701 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     5761 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaactgtcg tgccagctgc
     5821 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     5881 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     5941 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     6001 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     6061 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     6121 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     6181 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     6241 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     6301 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     6361 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     6421 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     6481 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     6541 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     6601 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     6661 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     6721 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     6781 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     6841 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     6901 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     6961 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     7021 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     7081 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     7141 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     7201 ca
//
