LOCUS       sequence-416264-        7833 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This Plasmid express M11 promoter driven SadCas9 specific gRNA and 
            EFS promoter driven NMS transactivation module fused to SadCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7833)
  AUTHORS   Mahata B, Cabrera A, Brenner DA, Guerra-Resendez RS, Li J, Goell J, 
            Wang K, Guo Y, Escobar M, Parthasarathy AK, Szadowski H, Bedford G, 
            Reed DR, Kim S, Hilton IB
  TITLE     Compact engineered human mechanosensitive transactivation modules 
            enable potent and versatile synthetic transcriptional control.
  JOURNAL   Nat Methods. 2023 Oct 9. doi: 10.1038/s41592-023-02036-1.
  PUBMED    37813990
REFERENCE   2  (bases 1 to 7833)
  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: "Nat 
            Methods. 2023 Oct 9. doi: 10.1038/s41592-023-02036-1."
COMMENT     Sequence Label: AAV-AIO-M11-gRNA-EFS-NMS-SadCas9
FEATURES             Location/Qualifiers
     source          1..7833
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(114..133)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      927..1515
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1416..1435
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1577..1706
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     1734..1753
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     promoter        1929..2140
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             6225..6272
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6273..6296
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(6359..6379)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     6565..6584
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     polyA_signal    6610..6658
                     /note="synthetic polyadenylation signal"
     repeat_region   6672..6812
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6672..6801
                     /label=AAV2 ITR
     rep_origin      6887..7342
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6974..6993)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7184..7205
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7359..7378)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7478..7500
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7538..7556)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7624..7728
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7729..7797,7798..7833,1..756)
                     /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"
ORIGIN
        1 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
       61 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
      121 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
      181 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
      241 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
      301 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
      361 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
      421 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
      481 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
      541 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
      601 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
      661 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
      721 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
      781 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
      841 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
      901 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
      961 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     1021 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     1081 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     1141 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     1201 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     1261 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     1321 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     1381 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     1441 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     1501 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     1561 ccttttgctc acatgtcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg
     1621 gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt
     1681 ggccaactcc atcactaggg gttcctcgcg tgtgtctaga catatttagc atgtcgctat
     1741 gtgttctggg aaacttgacc taagtgtaaa gttgagattt ccttcaggtt tatatagttc
     1801 tgtatgagac cactctttcc cgggtcttcg agaagacctg ttagagtact ctggaaacag
     1861 aatctactct aacaaggcaa aatgccgtgt ttatctcgtc aacttgttgg cgagattttt
     1921 ttgatatcgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     1981 caattgatcc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     2041 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     2101 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag tggtaccgcc accatgtcag
     2161 atgctttgta ctttgatgac tgcatgcagc ttttggcgca gacattcccg tttgtagatg
     2221 acaatgagtc gggaggtggt tcgggtggct ctggatcaag ccaggacatt gagcaagttt
     2281 gggaggagct attatccatt cctgagttac agtgtcttaa tattgaaaat gacaagctgg
     2341 ttgagtcggg atcctcaagc cagcagatgg acgacctgtt tgacattctc attcagagcg
     2401 gagaaatttc agcagatttc aaggagccgc catccctgcc agggaaggag aagccatccc
     2461 cgaaaacagt ctgtgggtcc cccctggcag cacagccatc accttctgct gagctccccc
     2521 aggctgcccc acctcctcca ggctcaccct ccctccctgg acgcctggag gacttcctgg
     2581 agagcagcac ggggctgccc ctgctgacca gtgggcatga cgggccagag cccctttccc
     2641 tcattgacga cctccatagc cagatgctga gcagcactgc catcctggac caccccccgt
     2701 cacccatgga cacctcggaa ttgcactttg ttcctgagcc cagcagcacc atgggcctgg
     2761 acctggctga tggccacctg gacagcatgg actggctgga gctgtcgtca ggtggtcccg
     2821 tgctgagcct agcccccctc agcaccacag cccccagcct cttctccaca gacttcctcg
     2881 atggccatga tttgcagctg cactgggatt cctcgggatc tgaagttcac ccttctagac
     2941 ttcagaccac agacaacctg ctccccatgt ctcctgagga gtttgacgag gtgtctcgga
     3001 tagtgggctc tgtagaattc gacagttcta gagcgctgag tggtggagga agtggcgggt
     3061 cagggtcgaa gcggaactac atcctgggcc tggccatcgg catcaccagc gtgggctacg
     3121 gcatcatcga ctacgagaca cgggacgtga tcgatgccgg cgtgcggctg ttcaaagagg
     3181 ccaacgtgga aaacaacgag ggtaggcgga gcaagagagg cgccagaagg ctgaagcggc
     3241 ggaggcggca tagaatccag agagtgaaga agctgctgtt cgactacaac ctgctgaccg
     3301 accacagcga gctgagcggc atcaacccct acgaggccag agtgaagggc ctgagccaga
     3361 agctgagcga ggaagagttc tctgccgccc tgctgcacct ggccaagaga agaggcgtgc
     3421 acaacgtgaa cgaggtggaa gaggacaccg gcaacgagct gtccaccaaa gagcagatca
     3481 gccggaacag caaggccctg gaagagaaat acgtggccga actgcagctg gaacggctga
     3541 agaaagacgg cgaagtgcgg ggcagcatca acagattcaa gaccagcgac tacgtgaaag
     3601 aagccaaaca gctgctgaag gtgcagaagg cctaccacca gctggaccag agcttcatcg
     3661 acacctacat cgacctgctg gaaacccggc ggacctacta tgagggacct ggcgagggca
     3721 gccccttcgg ctggaaggac atcaaagaat ggtacgagat gctgatgggc cactgcacct
     3781 acttccccga ggaactgcgg agcgtgaagt acgcctacaa cgccgacctg tacaacgccc
     3841 tgaacgacct gaacaatctc gtgatcacca gggacgagaa cgagaagctg gaatattacg
     3901 agaagttcca gatcatcgag aacgtgttca agcagaagaa gaagcccacc ctgaagcaga
     3961 tcgccaaaga aatcctcgtg aacgaagagg atattaaggg ctacagagtg accagcaccg
     4021 gcaagcccga gttcaccaac ctgaaggtgt accacgacat caaggacatt accgcccgga
     4081 aagagattat tgagaacgcc gagctgctgg atcagattgc caagatcctg accatctacc
     4141 agagcagcga ggacatccag gaagaactga ccaatctgaa ctccgagctg acccaggaag
     4201 agatcgagca gatctctaat ctgaagggct ataccggcac ccacaacctg agcctgaagg
     4261 ccatcaacct gatcctggac gagctgtggc acaccaacga caaccagatc gctatcttca
     4321 accggctgaa gctggtgccc aagaaggtgg acctgtccca gcagaaagag atccccacca
     4381 ccctggtgga cgacttcatc ctgagccccg tcgtgaagag aagcttcatc cagagcatca
     4441 aagtgatcaa cgccatcatc aagaagtacg gcctgcccaa cgacatcatt atcgagctgg
     4501 cccgcgagaa gaactccaag gacgcccaga aaatgatcaa cgagatgcag aagcggaacc
     4561 ggcagaccaa cgagcggatc gaggaaatca tccggaccac cggcaaagag aacgccaagt
     4621 acctgatcga gaagatcaag ctgcacgaca tgcaggaagg caagtgcctg tacagcctgg
     4681 aagccatccc tctggaagat ctgctgaaca accccttcaa ctatgaggtg gaccacatca
     4741 tccccagaag cgtgtccttc gacaacagct tcaacaacaa ggtgctcgtg aagcaggaag
     4801 aagccagcaa gaagggcaac cggaccccat tccagtacct gagcagcagc gacagcaaga
     4861 tcagctacga aaccttcaag aagcacatcc tgaatctggc caagggcaag ggcagaatca
     4921 gcaagaccaa gaaagagtat ctgctggaag aacgggacat caacaggttc tccgtgcaga
     4981 aagacttcat caaccggaac ctggtggata ccagatacgc caccagaggc ctgatgaacc
     5041 tgctgcggag ctacttcaga gtgaacaacc tggacgtgaa agtgaagtcc atcaatggcg
     5101 gcttcaccag ctttctgcgg cggaagtgga agtttaagaa agagcggaac aaggggtaca
     5161 agcaccacgc cgaggacgcc ctgatcattg ccaacgccga tttcatcttc aaagagtgga
     5221 agaaactgga caaggccaaa aaagtgatgg aaaaccagat gttcgaggaa aagcaggccg
     5281 agagcatgcc cgagatcgaa accgagcagg agtacaaaga gatcttcatc accccccacc
     5341 agatcaagca cattaaggac ttcaaggact acaagtacag ccaccgggtg gacaagaagc
     5401 ctaatagaga gctgattaac gacaccctgt actccacccg gaaggacgac aagggcaaca
     5461 ccctgatcgt gaacaatctg aacggcctgt acgacaagga caatgacaag ctgaaaaagc
     5521 tgatcaacaa gagccccgaa aagctgctga tgtaccacca cgacccccag acctaccaga
     5581 aactgaagct gattatggaa cagtacggcg acgagaagaa tcccctgtac aagtactacg
     5641 aggaaaccgg gaactacctg accaagtact ccaaaaagga caacggcccc gtgatcaaga
     5701 agattaagta ttacggcaac aaactgaacg cccatctgga catcaccgac gactacccca
     5761 acagcagaaa caaggtcgtg aagctgtccc tgaagcccta cagattcgac gtgtacctgg
     5821 acaatggcgt gtacaagttc gtgaccgtga agaatctgga tgtgatcaaa aaagaaaact
     5881 actacgaagt gaatagcaag tgctatgagg aagctaagaa gctgaagaag atcagcaacc
     5941 aggccgagtt tatcgcctcc ttctacaaca acgatctgat caagatcaac ggcgagctgt
     6001 atagagtgat cggcgtgaac aacgacctgc tgaaccggat cgaagtgaac atgatcgaca
     6061 tcacctaccg cgagtacctg gaaaacatga acgacaagag gccccccagg atcattaaga
     6121 caatcgcctc caagacccag agcattaaga agtacagcac agacattctg ggcaacctgt
     6181 atgaagtgaa atctaagaag caccctcaga tcatcaaaaa gggcaaaagg ccggcggcca
     6241 cgaaaaaggc cggccaggca aaaaagaaaa aggattacaa agacgatgac gataagtgag
     6301 ctagcaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     6361 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     6421 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttagttctt gccacggcgg
     6481 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     6541 attccgtggt ctagctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     6601 ataagctgca ataaaagatc tttattttca ttagatctgt gtgttggttt tttgtgtgat
     6661 cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     6721 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     6781 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     6841 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     6901 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     6961 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     7021 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     7081 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     7141 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     7201 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     7261 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     7321 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     7381 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     7441 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     7501 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     7561 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     7621 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     7681 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     7741 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     7801 ccagaaacgc tggtgaaagt aaaagatgct gaa
//
