LOCUS       sequence-174647-        9230 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for Cas9 from Geobacillus stearothermophilus with
            an N-Term MBP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9230)
  AUTHORS   Jennifer Doudna lab
  TITLE     GeoCas9 Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9230)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pET-MBP-Geo_st
FEATURES             Location/Qualifiers
     source          1..9230
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     98..117
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     351..368
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    383..522
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(448..467)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     608..630
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(624..815)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2273..2292
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2376..2395
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2376..2394
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2483..2505
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2525..2554
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2564..3664
                     /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     3638..3661
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3719..3739
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(7135..7153)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7149..7196
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7562..7567,7568..7584,7585..7590))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7617..7635)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7703..7807
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7808..7876,7877..8668)
                     /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(8026..8045)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(8839..9230,1..197)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
       61 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
      121 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
      181 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
      241 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
      301 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
      361 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
      421 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
      481 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
      541 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
      601 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
      661 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
      721 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
      781 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
      841 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
      901 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
      961 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     1021 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     1081 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     1141 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     1201 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     1261 agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg
     1321 ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca
     1381 gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc
     1441 cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga
     1501 ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg
     1561 aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag
     1621 ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg
     1681 cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca
     1741 tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg
     1801 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     1861 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     1921 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     1981 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     2041 agctgactgg gttgaaggct ctcaagggca tcggtcgacg ctctccctta tgcgactcct
     2101 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     2161 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     2221 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     2281 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     2341 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta tagggagacc
     2401 acaacggttt ccctctagtg ccggctccgg agagctcttt aattaagcgg ccgccctgca
     2461 ggactcgagt tctagaaata attttgttta actttaagaa ggagatatac atatgaaatc
     2521 ttctcaccat caccatcacc atcaccatca ccatggttct tctatgaaaa tcgaagaagg
     2581 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
     2641 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
     2701 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
     2761 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
     2821 ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc
     2881 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
     2941 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
     3001 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
     3061 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
     3121 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
     3181 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac
     3241 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
     3301 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
     3361 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     3421 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     3481 gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccactatgg aaaacgccca
     3541 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     3601 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     3661 gactaattcg agctcgaaca acaacaacaa taacaataac aacaacctcg ggatcgagga
     3721 aaacctgtac ttccaatcca atgcaatgcg ttataagatt ggcctggaca tcggtattac
     3781 ctctgttggt tgggcagtca tgaacctgga tatccctcgt atcgaagatc tgggcgtgcg
     3841 catttttgac cgtgcggaga acccgcagac cggtgaatct ctggctctgc cgcgtcgtct
     3901 ggcacgtagc gcacgccgcc gcctgcgtcg tcgtaaacac cgtctggagc gtattcgtcg
     3961 cctggttatt cgtgaaggca tcctgacgaa agaagaactg gataaactgt tcgaagaaaa
     4021 acacgagatc gacgtatggc agctgcgtgt agaagccctg gaccgtaagc tgaacaacga
     4081 cgaactggcg cgtgtcctgc tgcatctggc aaagcgtcgt ggcttcaaat ctaaccgtaa
     4141 atctgaacgc tccaataaag agaactccac tatgctgaaa catattgagg agaaccgtgc
     4201 aattctgtct agctaccgta ccgtgggcga aatgattgtt aaagacccga aattcgcact
     4261 gcataagcgt aacaaaggcg aaaactacac caacaccatt gcacgcgatg acctggaacg
     4321 tgaaatccgt ctgattttct ccaaacagcg cgaattcggc aacatgtctt gcaccgaaga
     4381 attcgaaaac gaatatatta ccatttgggc atctcagcgt ccggtggcgt ctaaagatga
     4441 tatcgaaaaa aaagtaggct tttgtacttt cgaaccgaag gaaaaacgtg cgccgaaagc
     4501 cacctatacc ttccagtctt ttatcgcgtg ggaacatatc aacaaactgc gtctgatttc
     4561 tccgtctggc gcccgcggcc tgaccgacga agaacgtcgt ctgctgtatg aacaagcatt
     4621 ccagaaaaac aaaattacct accacgatat tcgtaccctg ctgcatctgc cggacgacac
     4681 ctacttcaag ggcatcgttt acgatcgcgg tgaatctcgt aagcagaacg aaaacattcg
     4741 tttcctggaa ctggatgcat accaccagat ccgtaaagct gtagataaag tttacggcaa
     4801 gggtaaatcc agcagcttcc tgccgatcga ctttgatacc ttcggttacg cgctgaccct
     4861 gtttaaagac gatgcggata tccactctta cctgcgcaac gagtacgaac agaacggcaa
     4921 acgtatgcct aacctggcta acaaagttta cgataacgag ctgattgaag aactgctgaa
     4981 cctgtccttc actaaattcg gtcacctgtc tctgaaagct ctgcgttcca tcctgccgta
     5041 tatggaacag ggtgaagtct actcctccgc ttgtgaacgt gcaggctaca ccttcaccgg
     5101 tccgaaaaag aagcaaaaaa ctatgctgct gccgaacatc ccgccgattg cgaaccctgt
     5161 agtaatgcgt gcactgaccc aggcgcgcaa agtagtcaac gcgatcatca aaaagtacgg
     5221 cagcccggtt tccatccata tcgaactggc gcgcgacctg agccagactt ttgacgagcg
     5281 tcgtaaaact aaaaaggaac aggatgaaaa ccgtaaaaaa aacgaaaccg cgatccgcca
     5341 gctgatggaa tacggtctga ctctgaaccc tactggtcac gatattgtga agttcaagct
     5401 gtggtctgaa cagaacggtc gctgtgctta ctctctgcag ccgatcgaga tcgaacgtct
     5461 gctggagcca ggttacgttg aagtagatca tgtgatcccg tactcccgct ctctggatga
     5521 ttcttatacc aacaaagttc tggttctgac tcgcgaaaac cgtgagaaag gcaaccgcat
     5581 cccagctgaa tatctgggtg ttggcactga gcgttggcaa cagttcgaaa ccttcgtcct
     5641 gaccaataaa cagttctcta aaaagaaacg tgaccgtctg ctgcgtctgc actacgatga
     5701 aaacgaagag actgaattca aaaaccgtaa cctgaacgat actcgctaca tcagccgctt
     5761 cttcgcaaac ttcattcgtg aacacctgaa atttgcggaa tccgacgata aacagaaagt
     5821 ttataccgta aacggccgtg ttaccgccca cctgcgttct cgctgggagt tcaacaagaa
     5881 ccgtgaggaa agcgatctgc accacgctgt tgacgccgtt attgtggcgt gcaccacccc
     5941 aagcgatatc gctaaggtga ccgcattcta ccagcgtcgt gagcagaaca aggaactggc
     6001 caaaaaaacc gaaccgcatt ttccgcagcc gtggccgcac ttcgcggacg aactgcgtgc
     6061 tcgtctgtcc aaacatccta aagaaagcat caaagctctg aacctgggta actacgatga
     6121 ccaaaaactg gaatctctgc agccggtgtt tgtcagccgt atgccgaaac gttctgttac
     6181 tggcgctgcg caccaggaaa cgctgcgccg ttacgtgggc atcgacgaac gctccggtaa
     6241 aatccagacc gtagtaaaaa ccaaactgtc cgagattaaa ctggatgcat ccggccactt
     6301 cccaatgtac ggtaaagaat ccgatccacg cacttatgaa gccatccgcc agcgtctgct
     6361 ggagcataac aacgacccga aaaaggcatt ccaggagcct ctgtacaaac cgaaaaaaaa
     6421 cggcgaaccg ggcccggtaa tccgtactgt aaaaattatc gacacgaaaa accaggtgat
     6481 ccctctgaac gatggtaaaa ccgtggccta caattccaac atcgttcgcg tggacgtgtt
     6541 cgaaaaagat ggtaaatact actgtgtacc ggtgtatacc atggacatca tgaaaggcat
     6601 tctgccgaac aaagcgattg aaccgaacaa gccgtactct gaatggaaag aaatgaccga
     6661 agattacacg tttcgtttca gcctgtatcc gaacgacctg atccgcatcg aactgccgcg
     6721 tgaaaaaacc gttaaaaccg ctgcaggcga agaaattaac gtgaaagacg tgttcgttta
     6781 ctataaaacg atcgactccg caaacggcgg cctggaactg atttctcacg accaccgttt
     6841 ctctctgcgt ggcgttggct ctcgcaccct gaaacgtttc gagaaatatc aagttgatgt
     6901 tctgggtaac atctataaag tgcgtggcga gaaacgtgtc ggtctggcgt cctccgcaca
     6961 cagcaaacct ggcaaaacca ttcgtccact gcaatctact cgtgactaat aacattggaa
     7021 gtggataacg gatccgcgat cgcggcgcgc cacctggtgg ccggccggta ccacgcgtgc
     7081 gcgctgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     7141 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     7201 aaggaggaac tatatccgga tatccacagg acgggtgtgg tcgccatgat cgcgtagtcg
     7261 atagtggctc caagtagcga agcgagcagg actgggcggc ggccaaagcg gtcggacagt
     7321 gctccgagaa cgggtgcgca tagaaattgc atcaacgcat atagcgctag cagcacgcca
     7381 tagtgactgg cgatgctgtc ggaatggacg atatcccgca agaggcccgg cagtaccggc
     7441 ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca
     7501 ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg
     7561 cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct
     7621 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     7681 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     7741 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataac attgaaaaag
     7801 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     7861 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     7921 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     7981 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     8041 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     8101 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     8161 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     8221 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     8281 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     8341 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     8401 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     8461 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     8521 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     8581 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     8641 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     8701 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     8761 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     8821 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     8881 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     8941 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     9001 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     9061 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     9121 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     9181 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
//
