LOCUS       sequence-390941-        7228 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Nsp2-SmuCas9, and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7228)
  AUTHORS   Wei J, Hou L, Liu J, Wang Z, Gao S, Qi T, Gao S, Sun S, Wang Y
  TITLE     Closely related type II-C Cas9 orthologs recognize diverse PAMs.
  JOURNAL   Elife. 2022 Aug 12;11. pii: 77825. doi: 10.7554/eLife.77825.
  PUBMED    35959889
REFERENCE   2  (bases 1 to 7228)
  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: "Elife."; 
            date: "2022-08-12"; volume: "11. pii: 77825. doi"; pages: " 
            10.7554/eLife.77825"
COMMENT     Sequence Label: Nsp2-SmuCas9_AAV
FEATURES             Location/Qualifiers
     source          1..7228
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..21
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             28..75
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3286..3333
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3334..3423
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3430..3456
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     primer_bind     complement(3462..3481)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3527..3582
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   3595..3735
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3595..3724
                     /label=AAV2 ITR
     rep_origin      3810..4265
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3897..3916)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4107..4128
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4282..4301)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4401..4423
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4461..4479)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4547..4651
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4652..4720,4721..5512)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4870..4889)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5683..6271
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6172..6191
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6333..6462
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     misc_RNA        complement(6490..6582)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(6633..6881)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(6691..6710)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(6861..6881)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     regulatory      7217..7226
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 cctaagaaga agagaaaggt ggaagataaa cgcccagcag ctacaaagaa ggcaggtcaa
       61 gccaagaaaa agaaaaaaac ccctgcacac cggtacatcc tgggcctgga tctgggaatc
      121 gccagcgtgg gctgggccct ggtactggcc gacgagaatg aatatcctat cggcctgctg
      181 gattgcggcg tcagggtgtt cgagcgcgct gagatgccca aggacggaaa aagccttgcc
      241 gccacacgta gacaggcccg ctcgatcagg cggctgatcc gtagacgggc ccatagactg
      301 cttagactga gacggctgct gaagcgagaa ggcctcctgt ctgctgctga tttcgccgct
      361 gatggcctgc cggctgccat gcctgttaat acctggcagc tgagagtgga aggcctggac
      421 cgtaagctgg ccgaaaaaga gtgggccgcg gtgctgctgc atctggtcaa gcacagagga
      481 tacctgagcc agagaaagaa cgagatgcag accgaagata aggaactggg aaagctgctg
      541 gctggcgtgg ccgaaaatcg gcagctgctg gccgataacc ccgagcagta cagaacccct
      601 gccgagctgg ctgtgaagag gttcgccgtg cagagcggac acctgagaaa caagggtgga
      661 gattacgccc atacattcaa cagactagac ctgcagaagg aactgcacct cctgttcgcc
      721 aaacagcggg gactgggaaa tcctttcgcc tctgctgagc ttgagaacaa ggtcgactct
      781 ctgctgatga cccagagagc tgccctgcag gacggcgccg tgctgaaaat gctgggtcac
      841 tgcacctttg agccccaaga gtacaaggcc gccaaacaca cctacagcgc tgaaagattt
      901 atctggctga cccggctgaa caacctgcgg atcctgcaca acggagagga gcgggcgctg
      961 aacgagcacg agagaaagct gctgctggat gagccctaca agaaggccaa gctgacatac
     1021 aagcaggtgc ggaagatcct gaatctgccc gattctgccg tgttcaaggg cctgagatac
     1081 ggcaagagcg aggacggtct gaaggccgaa tctggcgccc tcatggaaat gaaagcctac
     1141 cacaagatcc ggaccgtgtt ggagaaggcc ggactgaagg gcgagtggga gaaccggatc
     1201 agaaatcccg aagttctgga cgccatcggc accgccttca gcctgtacaa aaccgacaac
     1261 gacatccaga ccgccctggc tgacctggca cttccagaaa aggtgctgga agccctgctg
     1321 aagggcatcg gcttcgacaa gttcatccag ctgagcctga gcgccctgcg aaagattctg
     1381 cctctgatgg aacaaggcct gcggtatgac gaagcctgta caaaggtgta cggcaaccac
     1441 tacggcgcta agattcagca gaatagcctg gtgctgcctg tgatcgatgg aaacgatatc
     1501 cggaatccag tggtgctgag aacactgacc caggccagaa aggtggtcaa cgccgtgatc
     1561 agaagatacg gcagccctct gagagtgcac atcgagacag ccagagaact gggaaaaagc
     1621 tgggacgata gaaaagagat tgagaagcgg caagaggaga accggaaaga gagagaaaga
     1681 gccgcagatg aattcaaaga atacttccct cactttgccg gcgaacctaa gggaagagat
     1741 atcctgaaaa tgagactgta caaccagcag cagggcaagt gcctgtacag cggcctgcct
     1801 ctggatctgt cccggctcaa cgagaagggc tacgtggaaa tcgaccacgc tctgcctttt
     1861 tctagaacct gggatgacag cttcaataac aaggtgctgg tgctgggcag cgaaaatcag
     1921 aaaaagagat cccagacacc ctacgagtac ctggacggcg cctccaacag cgagcggtgg
     1981 cgggaatttc aggccagagt ggccggctgc cactttcctt atgccaagaa gcaaagaatc
     2041 cagaccgcca aactgaacga gcaggacttc atcgaccgga acctgaacga caccaggtac
     2101 atcgcgcggt tcctgtgcgg cttcatcgag aacaacctgc atctggaagg caaggacaag
     2161 agaagagtgt tcgccagcaa cggccagatc accgcactgc tgcgcggact gtggggcctg
     2221 cgcaaggtgc gggaggagaa cgacagacac cacgccctgg acgccgtggt ggttgcctgc
     2281 agtaccgtga gcatgcagca aaagatcacc gaggccatga agcgcagaga aagcctggag
     2341 acagtgaaca cagagacagg cgaggtgaaa cagagaattc ctcaaccttg ggacttcttc
     2401 agacaggagg tgatgattcg cgtgttcagc gaccacccca gagaggaact cgccgaaaag
     2461 ctgaacagcc ggccacaggc cctgcacgag cacgtggccc ctctcttcgt gtccagagcc
     2521 cctaacagaa agaccaccgg ccagggccac ctggagacca ttaagagcgc caagaggctg
     2581 agcgaaaaca tcagcgtggt gaagaagcct ctggcatctc tcaaaacaaa ggacatcgag
     2641 aacatcgccg gctacaccgc caatagagag cctgagttct accaggctct gaagaagcgc
     2701 cttgccgagt tcaacgacga tcctaaaaag gcttttgccg agcccttcta caagcccacc
     2761 aagaacggcg agcagggccc tctggtgaaa tctgtgagag ttgaggatga ccgggtgaag
     2821 gtgggcctgg ccgtgcggca cggcatcaca gatacaaacg gcgacatggt gcgggtggac
     2881 gtgtacacca aagctggcaa aaactacctg gttcctgtgt acgtgtggca agtggctcag
     2941 ggcatcctgc caaatagagc cgtgaccagc ggaaaatccg aagccgactg ggacctgatc
     3001 gacgagagct tcgagttcaa gttctccctg agcagaggcg atctggttga aatgatcagc
     3061 aacaagggaa ggatctttgg ctactacaac ggcctggaca gagcgaatgg cagcatagga
     3121 atcagagagc acgacctgga aaagagcaaa ggcaaagatg gcgtgcatag agtaggcgtg
     3181 aaaaccgcca ccgccttcaa caagtaccac gtggaccctc tgggcaagga aatccacaga
     3241 tgcagcagcg agcctagacc taccctcaaa atcaagtcca agaaaaagcg gcccgcagca
     3301 accaagaagg caggacaggc caagaagaag aagtacccct atgacgtgcc tgactacgcc
     3361 gggtatccct acgacgtgcc tgattacgcc gggtcctatc cctacgacgt gccagattac
     3421 gctgcagctc cagcagcgaa gaaaaagaag ctggattaag atctttttcc ctctgccaaa
     3481 aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt
     3541 attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggcgg ccgcaggaac
     3601 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     3661 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     3721 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3781 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     3841 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     3901 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3961 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4021 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4081 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4141 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     4201 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     4261 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     4321 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4381 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4441 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4501 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4561 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4621 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4681 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4741 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4801 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4861 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4921 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4981 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5041 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     5101 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     5161 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     5221 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     5281 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5341 tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga
     5401 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5461 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5521 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5581 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5641 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5701 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5761 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5821 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5881 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5941 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6001 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6061 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6121 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6181 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6241 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6301 cctggccttt tgctggcctt ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct
     6361 cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag
     6421 cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggcctc tagagtttaa
     6481 acaaaaaaat aaacgatgcc ccttaaagca gaagctttaa ggggcagagc gttgcggcac
     6541 atcttttcag acggccttat tgtagcaacg gttctcattt cgtttccgaa atgagaaagg
     6601 gagctacaac tgaagagctg gaatgctctt ccggtgtttc gtcctttcca caagatatat
     6661 aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc cattttaaaa
     6721 cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca cgtattttgt
     6781 actaatatct ttgtgtttac agtcaaatta attctaatta tctctctaac agccttgtat
     6841 cgtatatgca aatatgaagg aatcatggga aataggccct cttcctgccc gaccttgacg
     6901 tcgactctag aatggaggcg gtactatgta gatgagaatt caggagcaaa ctgggaaaag
     6961 caactgcttc caaatatttg tgatttttac agtgtagttt tggaaaaact cttagcctac
     7021 caattcttct aagtgtttta aaatgtggga gccagtacac atgaagttat agagtgtttt
     7081 aatgaggctt aaatatttac cgtaactatg aaatgctacg catatcatgc tgttcaggct
     7141 ccgtggccac gcaactcata cttaagcaga cagtggttca aagttttttt cttccatttc
     7201 aggtgtcgtg aacaccgcca ccatggtg
//
