LOCUS       sequence-217552-        7937 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for His-MBP-Cas9 Alpha-Helical Lobe.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7937)
  AUTHORS   Wright AV, Sternberg SH, Taylor DW, Staahl BT, Bardales JA, Kornfeld
            JE, Doudna JA
  TITLE     Rational design of a split-Cas9 enzyme complex.
  JOURNAL   Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):2984-9. doi: 
            10.1073/pnas.1501698112. Epub 2015 Feb 23.
  PUBMED    25713377
REFERENCE   2  (bases 1 to 7937)
  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: 
            "10.1073/pnas.1501698112"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2015-03-10- 10"; volume: "112"; issue: "10"; pages: "2984-9"
COMMENT     Sequence Label: pAW-AlphaLobe
FEATURES             Location/Qualifiers
     source          1..7937
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..44
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        25..43
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             132..154
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             174..203
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             213..1313
                     /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     1287..1310
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1368..1388
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3491..3509)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3505..3552
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3918..3923,3924..3940,3941..3946))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3973..3991)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4059..4163
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4164..4232,4233..5024)
                     /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(4382..4401)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5195..5783
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5684..5703
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5937..5954
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5969..6108
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6034..6053)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6194..6216
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6210..6401)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     7859..7878
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atcgagatct cgatcccgcg aaattaatac gactcactat agggagacca caacggtttc
       61 cctctagtgc cggctccgga gagctcttta attaagcggc cgccctgcag gactcgagtt
      121 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgaaatct tctcaccatc
      181 accatcacca tcaccatcac catggttctt ctatgaaaat cgaagaaggt aaactggtaa
      241 tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga
      301 aagataccgg aattaaagtc accgttgagc atccggataa actggaagag aaattcccac
      361 aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg
      421 gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc
      481 tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg
      541 ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg ccaaaaacct
      601 gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt
      661 tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt
      721 tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcga
      781 aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg
      841 attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc
      901 cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga
      961 ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg
     1021 ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag
     1081 gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg
     1141 aagagttggc gaaagatcca cgtattgccg ccactatgga aaacgcccag aaaggtgaaa
     1201 tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact gcggtgatca
     1261 acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag actaattcga
     1321 gctcgaacaa caacaacaat aacaataaca acaacctcgg gatcgaggaa aacctgtact
     1381 tccaaggaga gacagcggaa gcgactcgtc tcaaacggac agctcgtaga aggtatacac
     1441 gtcggaagaa tcgtatttgt tatctacagg agattttttc aaatgagatg gcgaaagtag
     1501 atgatagttt ctttcatcga cttgaagagt cttttttggt ggaagaagac aagaagcatg
     1561 aacgtcatcc tatttttgga aatatagtag atgaagttgc ttatcatgag aaatatccaa
     1621 ctatctatca tctgcgaaaa aaattggtag attctactga taaagcggat ttgcgcttaa
     1681 tctatttggc cttagcgcat atgattaagt ttcgtggtca ttttttgatt gagggagatt
     1741 taaatcctga taatagtgat gtggacaaac tatttatcca gttggtacaa acctacaatc
     1801 aattatttga agaaaaccct attaacgcaa gtggagtaga tgctaaagcg attctttctg
     1861 cacgattgag taaatcaaga cgattagaaa atctcattgc tcagctcccc ggtgagaaga
     1921 aaaatggctt atttgggaat ctcattgctt tgtcattggg tttgacccct aattttaaat
     1981 caaattttga tttggcagaa gatgctaaat tacagctttc aaaagatact tacgatgatg
     2041 atttagataa tttattggcg caaattggag atcaatatgc tgatttgttt ttggcagcta
     2101 agaatttatc agatgctatt ttactttcag atatcctaag agtaaatact gaaataacta
     2161 aggctcccct atcagcttca atgattaaac gctacgatga acatcatcaa gacttgactc
     2221 ttttaaaagc tttagttcga caacaacttc cagaaaagta taaagaaatc ttttttgatc
     2281 aatcaaaaaa cggatatgca ggttatattg atgggggagc tagccaagaa gaattttata
     2341 aatttatcaa accaatttta gaaaaaatgg atggtactga ggaattattg gtgaaactaa
     2401 atcgtgaaga tttgctgcgc aagcaacgga cctttgacaa cggctctatt ccccatcaaa
     2461 ttcacttggg tgagctgcat gctattttga gaagacaaga agacttttat ccatttttaa
     2521 aagacaatcg tgagaagatt gaaaaaatct tgacttttcg aattccttat tatgttggtc
     2581 cattggcgcg tggcaatagt cgttttgcat ggatgactcg gaagtctgaa gaaacaatta
     2641 ccccatggaa ttttgaagaa gttgtcgata aaggtgcttc agctcaatca tttattgaac
     2701 gcatgacaaa ctttgataaa aatcttccaa atgaaaaagt actaccaaaa catagtttgc
     2761 tttatgagta ttttacggtt tataacgaat tgacaaaggt caaatatgtt actgaaggaa
     2821 tgcgaaaacc agcatttctt tcaggtgaac agaagaaagc cattgttgat ttactcttca
     2881 aaacaaatcg aaaagtaacc gttaagcaat taaaagaaga ttatttcaaa aaaatagaat
     2941 gttttgatag tgttgaaatt tcaggagttg aagatagatt taatgcttca ttaggtacct
     3001 accatgattt gctaaaaatt attaaagata aagatttttt ggataatgaa gaaaatgaag
     3061 atatcttaga ggatattgtt ttaacattga ccttatttga agatagggag atgattgagg
     3121 aaagacttaa aacatatgct cacctctttg atgataaggt gatgaaacag cttaaacgtc
     3181 gccgttatac tggttgggga cgtttgtctc gaaaattgat taatggtatt agggataagc
     3241 aatctggcaa aacaatatta gattttttga aatcagatgg ttttgccaat cgcaatttta
     3301 tgcagctgat ccatgatgat agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt
     3361 cttaataaca ttggaagtgg ataacggatc cgcgatcgcg gcgcgccacc tggtggccgg
     3421 ccggtaccac gcgtgcgcgc tgatccggct gctaacaaag cccgaaagga agctgagttg
     3481 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     3541 aggggttttt tgctgaaagg aggaactata tccggatatc cacaggacgg gtgtggtcgc
     3601 catgatcgcg tagtcgatag tggctccaag tagcgaagcg agcaggactg ggcggcggcc
     3661 aaagcggtcg gacagtgctc cgagaacggg tgcgcataga aattgcatca acgcatatag
     3721 cgctagcagc acgccatagt gactggcgat gctgtcggaa tggacgatat cccgcaagag
     3781 gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag
     3841 gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa
     3901 ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt
     3961 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     4021 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     4081 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     4141 aataacattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     4201 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     4261 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     4321 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     4381 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     4441 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     4501 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4561 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4621 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4681 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     4741 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4801 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4861 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4921 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4981 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     5041 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5101 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5161 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5221 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5281 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5341 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5401 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5461 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5521 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5581 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5641 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5701 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5761 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5821 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5881 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5941 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     6001 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     6061 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     6121 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     6181 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     6241 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     6301 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     6361 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     6421 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     6481 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     6541 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     6601 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     6661 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     6721 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     6781 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     6841 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc
     6901 gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca
     6961 ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg
     7021 aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg
     7081 cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc
     7141 tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg
     7201 taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca
     7261 gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga
     7321 aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca
     7381 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     7441 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     7501 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     7561 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     7621 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgacgctct cccttatgcg
     7681 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     7741 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     7801 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     7861 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     7921 tgcgtccggc gtagagg
//
