LOCUS       sequence-176992-        9263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for Cas9 from Geobacillus stearothermophilus with
            an N-Term MBP and SV40 NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9263)
  AUTHORS   Jennifer Doudna lab
  TITLE     GeoCas9 Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9263)
  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            "Unpublished"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-NLS-Geo_st
FEATURES             Location/Qualifiers
     source          1..9263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     44..63
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     147..166
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        147..165
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             254..276
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             296..325
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             335..1435
                     /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     1409..1432
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1490..1510
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1514..1534
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4939..4957)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4953..5000
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5366..5371,5372..5388,5389..5394))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5421..5439)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5507..5611
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5612..5680,5681..6472)
                     /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(5830..5849)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6643..7231
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7132..7151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7385..7402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7417..7556
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7482..7501)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7642..7664
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7658..7849)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
       61 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
      121 ggatcgagat ctcgatcccg cgaaattaat acgactcact atagggagac cacaacggtt
      181 tccctctagt gccggctccg gagagctctt taattaagcg gccgccctgc aggactcgag
      241 ttctagaaat aattttgttt aactttaaga aggagatata catatgaaat cttctcacca
      301 tcaccatcac catcaccatc accatggttc ttctatgaaa atcgaagaag gtaaactggt
      361 aatctggatt aacggcgata aaggctataa cggtctcgct gaagtcggta agaaattcga
      421 gaaagatacc ggaattaaag tcaccgttga gcatccggat aaactggaag agaaattccc
      481 acaggttgcg gcaactggcg atggccctga cattatcttc tgggcacacg accgctttgg
      541 tggctacgct caatctggcc tgttggctga aatcaccccg gacaaagcgt tccaggacaa
      601 gctgtatccg tttacctggg atgccgtacg ttacaacggc aagctgattg cttacccgat
      661 cgctgttgaa gcgttatcgc tgatttataa caaagatctg ctgccgaacc cgccaaaaac
      721 ctgggaagag atcccggcgc tggataaaga actgaaagcg aaaggtaaga gcgcgctgat
      781 gttcaacctg caagaaccgt acttcacctg gccgctgatt gctgctgacg ggggttatgc
      841 gttcaagtat gaaaacggca agtacgacat taaagacgtg ggcgtggata acgctggcgc
      901 gaaagcgggt ctgaccttcc tggttgacct gattaaaaac aaacacatga atgcagacac
      961 cgattactcc atcgcagaag ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg
     1021 cccgtgggca tggtccaaca tcgacaccag caaagtgaat tatggtgtaa cggtactgcc
     1081 gaccttcaag ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc
     1141 cgccagtccg aacaaagagc tggcaaaaga gttcctcgaa aactatctgc tgactgatga
     1201 aggtctggaa gcggttaata aagacaaacc gctgggtgcc gtagcgctga agtcttacga
     1261 ggaagagttg gcgaaagatc cacgtattgc cgccactatg gaaaacgccc agaaaggtga
     1321 aatcatgccg aacatcccgc agatgtccgc tttctggtat gccgtgcgta ctgcggtgat
     1381 caacgccgcc agcggtcgtc agactgtcga tgaagccctg aaagacgcgc agactaattc
     1441 gagctcgaac aacaacaaca ataacaataa caacaacctc gggatcgagg aaaacctgta
     1501 cttccaatcc atgcccaaga agaagaggaa ggtgggcggt tcgaatgcaa tgcgttataa
     1561 gattggcctg gacatcggta ttacctctgt tggttgggca gtcatgaacc tggatatccc
     1621 tcgtatcgaa gatctgggcg tgcgcatttt tgaccgtgcg gagaacccgc agaccggtga
     1681 atctctggct ctgccgcgtc gtctggcacg tagcgcacgc cgccgcctgc gtcgtcgtaa
     1741 acaccgtctg gagcgtattc gtcgcctggt tattcgtgaa ggcatcctga cgaaagaaga
     1801 actggataaa ctgttcgaag aaaaacacga gatcgacgta tggcagctgc gtgtagaagc
     1861 cctggaccgt aagctgaaca acgacgaact ggcgcgtgtc ctgctgcatc tggcaaagcg
     1921 tcgtggcttc aaatctaacc gtaaatctga acgctccaat aaagagaact ccactatgct
     1981 gaaacatatt gaggagaacc gtgcaattct gtctagctac cgtaccgtgg gcgaaatgat
     2041 tgttaaagac ccgaaattcg cactgcataa gcgtaacaaa ggcgaaaact acaccaacac
     2101 cattgcacgc gatgacctgg aacgtgaaat ccgtctgatt ttctccaaac agcgcgaatt
     2161 cggcaacatg tcttgcaccg aagaattcga aaacgaatat attaccattt gggcatctca
     2221 gcgtccggtg gcgtctaaag atgatatcga aaaaaaagta ggcttttgta ctttcgaacc
     2281 gaaggaaaaa cgtgcgccga aagccaccta taccttccag tcttttatcg cgtgggaaca
     2341 tatcaacaaa ctgcgtctga tttctccgtc tggcgcccgc ggcctgaccg acgaagaacg
     2401 tcgtctgctg tatgaacaag cattccagaa aaacaaaatt acctaccacg atattcgtac
     2461 cctgctgcat ctgccggacg acacctactt caagggcatc gtttacgatc gcggtgaatc
     2521 tcgtaagcag aacgaaaaca ttcgtttcct ggaactggat gcataccacc agatccgtaa
     2581 agctgtagat aaagtttacg gcaagggtaa atccagcagc ttcctgccga tcgactttga
     2641 taccttcggt tacgcgctga ccctgtttaa agacgatgcg gatatccact cttacctgcg
     2701 caacgagtac gaacagaacg gcaaacgtat gcctaacctg gctaacaaag tttacgataa
     2761 cgagctgatt gaagaactgc tgaacctgtc cttcactaaa ttcggtcacc tgtctctgaa
     2821 agctctgcgt tccatcctgc cgtatatgga acagggtgaa gtctactcct ccgcttgtga
     2881 acgtgcaggc tacaccttca ccggtccgaa aaagaagcaa aaaactatgc tgctgccgaa
     2941 catcccgccg attgcgaacc ctgtagtaat gcgtgcactg acccaggcgc gcaaagtagt
     3001 caacgcgatc atcaaaaagt acggcagccc ggtttccatc catatcgaac tggcgcgcga
     3061 cctgagccag acttttgacg agcgtcgtaa aactaaaaag gaacaggatg aaaaccgtaa
     3121 aaaaaacgaa accgcgatcc gccagctgat ggaatacggt ctgactctga accctactgg
     3181 tcacgatatt gtgaagttca agctgtggtc tgaacagaac ggtcgctgtg cttactctct
     3241 gcagccgatc gagatcgaac gtctgctgga gccaggttac gttgaagtag atcatgtgat
     3301 cccgtactcc cgctctctgg atgattctta taccaacaaa gttctggttc tgactcgcga
     3361 aaaccgtgag aaaggcaacc gcatcccagc tgaatatctg ggtgttggca ctgagcgttg
     3421 gcaacagttc gaaaccttcg tcctgaccaa taaacagttc tctaaaaaga aacgtgaccg
     3481 tctgctgcgt ctgcactacg atgaaaacga agagactgaa ttcaaaaacc gtaacctgaa
     3541 cgatactcgc tacatcagcc gcttcttcgc aaacttcatt cgtgaacacc tgaaatttgc
     3601 ggaatccgac gataaacaga aagtttatac cgtaaacggc cgtgttaccg cccacctgcg
     3661 ttctcgctgg gagttcaaca agaaccgtga ggaaagcgat ctgcaccacg ctgttgacgc
     3721 cgttattgtg gcgtgcacca ccccaagcga tatcgctaag gtgaccgcat tctaccagcg
     3781 tcgtgagcag aacaaggaac tggccaaaaa aaccgaaccg cattttccgc agccgtggcc
     3841 gcacttcgcg gacgaactgc gtgctcgtct gtccaaacat cctaaagaaa gcatcaaagc
     3901 tctgaacctg ggtaactacg atgaccaaaa actggaatct ctgcagccgg tgtttgtcag
     3961 ccgtatgccg aaacgttctg ttactggcgc tgcgcaccag gaaacgctgc gccgttacgt
     4021 gggcatcgac gaacgctccg gtaaaatcca gaccgtagta aaaaccaaac tgtccgagat
     4081 taaactggat gcatccggcc acttcccaat gtacggtaaa gaatccgatc cacgcactta
     4141 tgaagccatc cgccagcgtc tgctggagca taacaacgac ccgaaaaagg cattccagga
     4201 gcctctgtac aaaccgaaaa aaaacggcga accgggcccg gtaatccgta ctgtaaaaat
     4261 tatcgacacg aaaaaccagg tgatccctct gaacgatggt aaaaccgtgg cctacaattc
     4321 caacatcgtt cgcgtggacg tgttcgaaaa agatggtaaa tactactgtg taccggtgta
     4381 taccatggac atcatgaaag gcattctgcc gaacaaagcg attgaaccga acaagccgta
     4441 ctctgaatgg aaagaaatga ccgaagatta cacgtttcgt ttcagcctgt atccgaacga
     4501 cctgatccgc atcgaactgc cgcgtgaaaa aaccgttaaa accgctgcag gcgaagaaat
     4561 taacgtgaaa gacgtgttcg tttactataa aacgatcgac tccgcaaacg gcggcctgga
     4621 actgatttct cacgaccacc gtttctctct gcgtggcgtt ggctctcgca ccctgaaacg
     4681 tttcgagaaa tatcaagttg atgttctggg taacatctat aaagtgcgtg gcgagaaacg
     4741 tgtcggtctg gcgtcctccg cacacagcaa acctggcaaa accattcgtc cactgcaatc
     4801 tactcgtgac taataacatt ggaagtggat aacggatccg cgatcgcggc gcgccacctg
     4861 gtggccggcc ggtaccacgc gtgcgcgctg atccggctgc taacaaagcc cgaaaggaag
     4921 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     4981 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatcca caggacgggt
     5041 gtggtcgcca tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag caggactggg
     5101 cggcggccaa agcggtcgga cagtgctccg agaacgggtg cgcatagaaa ttgcatcaac
     5161 gcatatagcg ctagcagcac gccatagtga ctggcgatgc tgtcggaatg gacgatatcc
     5221 cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg
     5281 gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag
     5341 caatttaact gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga
     5401 gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     5461 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5521 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     5581 aatgcttcaa taacattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     5641 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     5701 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     5761 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     5821 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     5881 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     5941 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     6001 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     6061 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     6121 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     6181 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     6241 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     6301 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     6361 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     6421 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     6481 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     6541 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     6601 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6661 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6721 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6781 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6841 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6901 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6961 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     7021 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     7081 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     7141 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     7201 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     7261 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     7321 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     7381 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     7441 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     7501 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     7561 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     7621 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     7681 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     7741 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     7801 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     7861 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     7921 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     7981 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     8041 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     8101 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     8161 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     8221 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     8281 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga
     8341 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
     8401 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
     8461 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
     8521 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt
     8581 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt
     8641 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg
     8701 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat
     8761 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc
     8821 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     8881 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     8941 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     9001 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     9061 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc
     9121 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     9181 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     9241 tgccaccata cccacgccga aac
//
