LOCUS       sequence-416261-        6314 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This Plasmid express U6 promoter driven SpdCas9 specific gRNA and 
            hSyn promoter driven MCP-MSN transactivation module.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6314)
  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 6314)
  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-U6-Agrp-(mouse)-MS2gRNA-hSyn-MCP-MSN
FEATURES             Location/Qualifiers
     source          1..6314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        154..402
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     154..174
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     325..344
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        441..459
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        511..529
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        588..1035
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      1058..1067
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1067..1453
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             1508..1528
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2441..3029
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2494..2514)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2912..2923)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3061..3537
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3260..3279)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3577..3717
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3577..3706
                     /label=AAV2 ITR
     rep_origin      3792..4247
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3879..3898)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4089..4110
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4264..4283)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4383..4405
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4443..4461)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4529..4633
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4634..4702,4703..5494)
                     /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(4852..4871)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5665..6253
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6154..6173
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgtgtct agagagggcc tatttcccat gattccttca
      181 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
      241 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
      301 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
      361 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgagtaagg actgcacatt
      421 tgtgttttag agctaggcca acatgaggat cacccatgtc tgcagggcct agcaagttaa
      481 aataaggcta gtccgttatc aacttggcca acatgaggat cacccatgtc tgcagggcca
      541 agtggcaccg agtcggtgct ttttttctgc agagggccct gcgtatgagt gcaagtgggt
      601 tttaggacca ggatgaggcg gggtgggggt gcctacctga cgaccgaccc cgacccactg
      661 gacaagcacc caacccccat tccccaaatt gcgcatcccc tatcagagag ggggagggga
      721 aacaggatgc ggcgaggcgc gtgcgcactg ccagcttcag caccgcggac agtgccttcg
      781 cccccgcctg gcggcgcgcg ccaccgccgc ctcagcactg aaggcgcgct gacgtcactc
      841 gccggtcccc cgcaaactcc ccttcccggc caccttggtc gcgtccgcgc cgccgccggc
      901 ccagccggac cgcaccacgc gaggcgcgag ataggggggc acgggcgcga ccatctgcgc
      961 tgcggcgccg gcgactcagc gctgcctcag tctgcggtgg gcagcggagg agtcgtgtcg
     1021 tgcctgagag cgcagtcgag acgtgggtac cagatctgcc accatggctt caaactttac
     1081 tcagttcgtg ctcgtggaca atggtgggac aggggatgtg acagtggctc cttctaattt
     1141 cgctaatggg gtggcagagt ggatcagctc caactcacgg agccaggcct acaaggtgac
     1201 atgcagcgtc aggcagtcta gtgcccagaa gagaaagtat accatcaagg tggaggtccc
     1261 caaagtggct acccagacag tgggcggagt cgaactgcct gtcgccgctt ggaggtccta
     1321 cctgaacatg gagctcacta tcccaatttt cgctaccaat tctgactgtg aactcatcgt
     1381 gaaggcaatg caggggctcc tcaaagacgg taatcctatc ccttccgcca tcgccgctaa
     1441 ctcaggtatc tacagcgctg gaggaggtgg aagcggagga ggaggaagcg gaggaggagg
     1501 tagcggacct aagaaaaaga ggaaggtggc ggccgctgga tcctcctcaa gccagcagat
     1561 ggacgacctg tttgacattc tcattcagag cggagaaatt tcagcagatt tcaaggagcc
     1621 gccatccctg ccagggaagg agaagccatc cccgaagaca gtctgtgggt cccccctggc
     1681 agcacagcca tcaccttctg ctgagctccc ccaggctgcc ccacctcctc caggctcacc
     1741 ctccctccct ggacgcctgg aggacttcct ggagagcagc acggggctgc ccctgctgac
     1801 cagtgggcat gacgggccag agcccctttc cctcattgac gacctccata gccagatgct
     1861 gagcagcact gccatcctgg accacccccc gtcacccatg gacacctcgg aattgcactt
     1921 tgttcctgag cccagcagca ccatgggcct ggacctggct gatggccacc tggacagcat
     1981 ggactggctg gagctgtcgt caggtggtcc cgtgctgagc ctagcccccc tcagcaccac
     2041 agcccccagc ctcttctcca cagacttcct cgatggccat gatttgcagc tgcactggga
     2101 ttcctcggga tctgaagttc acccttctag acttcagacc acagacaacc tgctccccat
     2161 gtctcctgag gagtttgacg aggtgtctcg gatagtgggc tctgtagaat tcgacagttc
     2221 gggatcagat gctttgtact ttgatgactg catgcagctt ttggcgcaga cattcccgtt
     2281 tgtagatgac aatgagtcgg gaggtggttc gggtggctct ggatcaagcc aggacattga
     2341 gcaagtttgg gaggagctat tatccattcc tgagttacag tgtcttaata ttgaaaatga
     2401 caagctggtt gagtgagaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa
     2461 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     2521 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     2581 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     2641 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     2701 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     2761 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     2821 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat
     2881 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     2941 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     3001 tcggatctcc ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac
     3061 gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca
     3121 gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc
     3181 ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca
     3241 acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg
     3301 ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg
     3361 ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg
     3421 ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct
     3481 tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg
     3541 attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg
     3601 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     3661 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc
     3721 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
     3781 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3841 cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc
     3901 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3961 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     4021 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4081 ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc
     4141 ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta
     4201 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
     4261 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     4321 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4381 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     4441 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     4501 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4561 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     4621 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     4681 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     4741 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     4801 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     4861 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     4921 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     4981 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5041 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5101 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     5161 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     5221 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     5281 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     5341 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     5401 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     5461 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     5521 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5581 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5641 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5701 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5761 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     5821 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5881 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5941 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6001 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6061 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6121 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6181 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6241 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6301 ttttgctcac atgt
//
