LOCUS       sequence-233638-        8691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nme2Cas9 bacterial expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8691)
  AUTHORS   Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y, 
            Gallant J, Xue W, Rivera-Perez JA, Sontheimer EJ
  TITLE     A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo 
            Genome Editing.
  JOURNAL   Mol Cell. 2018 Dec 18. pii: S1097-2765(18)31033-5. doi: 
            10.1016/j.molcel.2018.12.003.
  PUBMED    30581144
REFERENCE   2  (bases 1 to 8691)
  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: "Mol Cell. 
            2018 Dec 18. pii: S1097-2765(18)31033-5. doi: 
            10.1016/j.molcel.2018.12.003."
COMMENT     Sequence Label: Nme2Cas9_pMCSG7
FEATURES             Location/Qualifiers
     source          1..8691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     963..982
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1002..1079)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1285..1304
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1392..1411
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1392..1410
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1411..1435
                     /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             1450..1472
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1483..1500
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1525..1545
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1546..1566
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4864..4890
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             4903..4950
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5019..5036
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5089..5107)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5103..5150
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(5274..5293)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5460..5563
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5564..5632,5633..6424)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5782..5801)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6595..7183
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7084..7103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7337..7354
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7369..7508
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7434..7453)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7594..7616
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7610..7801)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(8610..8691,1..1001))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
       61 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
      121 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
      181 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
      241 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
      301 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
      361 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
      421 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
      481 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
      541 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
      601 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
      661 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
      721 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
      781 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
      841 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
      901 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
      961 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     1021 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     1081 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     1141 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     1201 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     1261 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     1321 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatctcga
     1381 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     1441 agaaataatt ttgtttaact ttaagaagga gatatacata tgcaccatca tcatcatcat
     1501 tcttctggtg tagatctggg taccgagaac ctgtacttcc aatcccctaa gaagaagaga
     1561 aaggtgaatg ccatggccgc cttcaagcct aacccaatca attacatcct gggactggac
     1621 atcggaatcg catccgtggg atgggctatg gtggagatcg acgaggagga gaatcctatc
     1681 cggctgatcg atctgggcgt gagagtgttt gagagggccg aggtgccaaa gaccggcgat
     1741 tctctggcta tggcccggag actggcacgg agcgtgaggc gcctgacacg gagaagggca
     1801 cacaggctgc tgagggcacg ccggctgctg aagagagagg gcgtgctgca ggcagcagac
     1861 ttcgatgaga atggcctgat caagagcctg ccaaacaccc cctggcagct gagagcagcc
     1921 gccctggaca ggaagctgac accactggag tggtctgccg tgctgctgca cctgatcaag
     1981 caccgcggct acctgagcca gcggaagaac gagggagaga cagcagacaa ggagctgggc
     2041 gccctgctga agggagtggc caacaatgcc cacgccctgc agaccggcga tttcaggaca
     2101 cctgccgagc tggccctgaa taagtttgag aaggagtccg gccacatcag aaaccagagg
     2161 ggcgactata gccacacctt ctcccgcaag gatctgcagg ccgagctgat cctgctgttc
     2221 gagaagcaga aggagtttgg caatccacac gtgagcggag gcctgaagga gggaatcgag
     2281 accctgctga tgacacagag gcctgccctg tccggcgacg cagtgcagaa gatgctggga
     2341 cactgcacct tcgagcctgc agagccaaag gccgccaaga acacctacac agccgagcgg
     2401 tttatctggc tgacaaagct gaacaatctg agaatcctgg agcagggatc cgagaggcca
     2461 ctgaccgaca cagagagggc caccctgatg gatgagcctt accggaagtc taagctgaca
     2521 tatgcccagg ccagaaagct gctgggcctg gaggacaccg ccttctttaa gggcctgaga
     2581 tacggcaagg ataatgccga ggcctccaca ctgatggaga tgaaggccta tcacgccatc
     2641 tctcgcgccc tggagaagga gggcctgaag gacaagaagt cccccctgaa cctgagctcc
     2701 gagctgcagg atgagatcgg caccgccttc tctctgttta agaccgacga ggatatcaca
     2761 ggccgcctga aggacagggt gcagcctgag atcctggagg ccctgctgaa gcacatctct
     2821 ttcgataagt ttgtgcagat cagcctgaag gccctgagaa ggatcgtgcc actgatggag
     2881 cagggcaagc ggtacgacga ggcctgcgcc gagatctacg gcgatcacta tggcaagaag
     2941 aacacagagg agaagatcta tctgccccct atccctgccg acgagatcag aaatcctgtg
     3001 gtgctgaggg ccctgtccca ggcaagaaaa gtgatcaacg gagtggtgcg ccggtacgga
     3061 tctccagccc ggatccacat cgagaccgcc agagaagtgg gcaagagctt caaggaccgg
     3121 aaggagatcg agaagagaca ggaggagaat cgcaaggatc gggagaaggc cgccgccaag
     3181 tttagggagt acttccctaa ctttgtgggc gagccaaagt ctaaggacat cctgaagctg
     3241 cgcctgtacg agcagcagca cggcaagtgt ctgtatagcg gcaaggagat caatctggtg
     3301 cggctgaacg agaagggcta tgtggagatc gatcacgccc tgcctttctc cagaacctgg
     3361 gacgattctt ttaacaataa ggtgctggtg ctgggcagcg agaaccagaa taagggcaat
     3421 cagacaccat acgagtattt caatggcaag gacaactcca gggagtggca ggagttcaag
     3481 gcccgcgtgg agacctctag atttcccagg agcaagaagc agcggatcct gctgcagaag
     3541 ttcgacgagg atggctttaa ggagtgcaac ctgaatgaca ccagatacgt gaaccggttc
     3601 ctgtgccagt ttgtggccga tcacatcctg ctgaccggca agggcaagag aagggtgttc
     3661 gcctctaatg gccagatcac aaacctgctg aggggatttt ggggactgag gaaggtgcgg
     3721 gcagagaatg acagacacca cgcactggat gcagtggtgg tggcatgcag caccgtggca
     3781 atgcagcaga agatcacaag attcgtgagg tataaggaga tgaacgcctt tgacggcaag
     3841 accatcgata aggagacagg caaggtgctg caccagaaga cccacttccc ccagccttgg
     3901 gagttctttg cccaggaagt gatgatccgg gtgttcggca agccagacgg caagcctgag
     3961 tttgaggagg ccgatacccc agagaagctg aggacactgc tggcagagaa gctgtctagc
     4021 aggccagagg cagtgcacga gtacgtgacc ccactgttcg tgtccagggc acccaatcgg
     4081 aagatgtctg gcgcccacaa ggacacactg agaagcgcca agaggtttgt gaagcacaac
     4141 gagaagatct ccgtgaagag agtgtggctg accgagatca agctggccga tctggagaac
     4201 atggtgaatt acaagaacgg cagggagatc gagctgtatg aggccctgaa ggcaaggctg
     4261 gaggcctacg gaggaaatgc caagcaggcc ttcgacccaa aggataaccc cttttataag
     4321 aagggaggac agctggtgaa ggccgtgcgg gtggagaaga cccaggagag cggcgtgctg
     4381 ctgaataaga agaacgccta cacaatcgcc gacaatggcg atatggtgag agtggacgtg
     4441 ttctgtaagg tggataagaa gggcaagaat cagtacttta tcgtgcctat ctatgcctgg
     4501 caggtggccg agaacatcct gccagacatc gattgcaagg gctacagaat cgacgatagc
     4561 tatacattct gtttttccct gcacaagtat gacctgatcg ccttccagaa ggatgagaag
     4621 tccaaggtgg agtttgccta ctatatcaat tgcgactcct ctaacggcag gttctacctg
     4681 gcctggcacg ataagggcag caaggagcag cagtttcgca tctccaccca gaatctggtg
     4741 ctgatccaga agtatcaggt gaacgagctg ggcaaggaga tcaggccatg tcggctgaag
     4801 aagcgcccac ccgtgcgggg cgggggtggt tcaggtggag gagggtcagg cggagggggt
     4861 tcgccagcag cgaagaaaaa gaagctggat ggcgggggat ctaagcgccc agccgctaca
     4921 aaaaaagcgg gacaggcgaa gaagaagaag taaaacattg gaagtggata acggatccga
     4981 attcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac caccactgag
     5041 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     5101 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     5161 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     5221 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     5281 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     5341 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     5401 ttagggtgat ggttcacatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     5461 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5521 aataaccctg ataaatgctt caataatttg aaaaaggaag agtatgagta ttcaacattt
     5581 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5641 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5701 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5761 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5821 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5881 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5941 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6001 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6061 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac
     6121 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6181 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6241 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6301 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6361 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6421 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6481 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6541 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6601 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6661 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6721 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     6781 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6841 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6901 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6961 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7021 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7081 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7141 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7201 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ttgcgttatc
     7261 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7321 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     7381 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     7441 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     7501 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     7561 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     7621 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     7681 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     7741 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     7801 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     7861 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     7921 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     7981 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     8041 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     8101 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     8161 tgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     8221 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     8281 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     8341 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     8401 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     8461 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     8521 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     8581 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     8641 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg c
//
