LOCUS       sequence-469476-        6764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mCherry, sesRNA in mammalian cells, with msFlag and 
            tTA2 as efRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6764)
  AUTHORS   Qian Y, Li J, Zhao S, Matthews EA, Adoff M, Zhong W, An X, Yeo M, 
            Park C, Yang X, Wang BS, Southwell DG, Huang ZJ
  TITLE     Programmable RNA sensing for cell monitoring and manipulation.
  JOURNAL   Nature. 2022 Oct;610(7933):713-721. doi: 10.1038/s41586-022-05280-1.
            Epub 2022 Oct 5.
  PUBMED    36198803
REFERENCE   2  (bases 1 to 6764)
  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"; doi: 
            "10.1038/s41586-022-05280-1"; journalName: "Nature"; date: 
            "2022-10"; volume: "610"; issue: "7933"; pages: "713-721"
COMMENT     Sequence Label: pAAV-hSyn-mCherry-VGAT sesRNA-2a-msFlag-2a-tTA...
FEATURES             Location/Qualifiers
     source          1..6764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..46
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   188..317
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        362..809
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      829..838
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             835..1542
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(980..998)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1248..1267
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1858..1914
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1918..1941
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1945..1968
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1975..1998
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2527..2550
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2557..2580
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2593..2616
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2626..2649
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2860..2883
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2890..2913
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2917..2940
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2953..3006
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(3016..3630,3631..3636,3637..3672,3673..3675,3676..3711,
                     3712..3714,3715..3750,3751..3759)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=tTA-Advanced
                     /note="In the Tet-Off(R) system, tTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTD
                     SMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPAD
                     ALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(3834..3854)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4214..4354
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4214..4343
                     /label=AAV2 ITR
     rep_origin      4429..4884
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4516..4535)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4726..4747
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4901..4920)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5020..5042
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5080..5098)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5166..5270
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5271..5339,5340..6131)
                     /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(5489..5508)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6302..6764,1..126)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
       61 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
      121 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
      181 cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc
      241 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
      301 catcactagg ggttcctgcg gccgcacgcg tgtgtctaga ctgcagaggg ccctgcgtat
      361 gagtgcaagt gggttttagg accaggatga ggcggggtgg gggtgcctac ctgacgaccg
      421 accccgaccc actggacaag cacccaaccc ccattcccca aattgcgcat cccctatcag
      481 agagggggag gggaaacagg atgcggcgag gcgcgtgcgc actgccagct tcagcaccgc
      541 ggacagtgcc ttcgcccccg cctggcggcg cgcgccaccg ccgcctcagc actgaaggcg
      601 cgctgacgtc actcgccggt cccccgcaaa ctccccttcc cggccacctt ggtcgcgtcc
      661 gcgccgccgc cggcccagcc ggaccgcacc acgcgaggcg cgagataggg gggcacgggc
      721 gcgaccatct gcgctgcggc gccggcgact cagcgctgcc tcagtctgcg gtgggcagcg
      781 gaggagtcgt gtcgtgcctg agagcgcagt cgagaaggta ccggatccgc caccatggtg
      841 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
      901 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
      961 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     1021 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     1081 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     1141 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     1201 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     1261 aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
     1321 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     1381 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     1441 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     1501 gagggccgcc actccaccgg cggcatggac gagctgtaca aggaattccc tggtccttgg
     1561 agccagaggg tggcagagga gcgccgccgc gctgataatg aatgtctccc tcgacgggag
     1621 cttctgcgcc ctcgtctccg cagggctcgc cttccgattt caggatgtcc atctgcaggc
     1681 cctggcgatg ctcaaagtcg agatcgtcgc agtgcgcgaa gcccaccgct tcctcatcgg
     1741 tagccgcctg aaaccccatc ctagcaaaca tgccgctcac cttggcctgg gacttgttgg
     1801 acacggaggt ggccacattg gtcagcttgc tgcggagcag ggtggccagg cgcgccagct
     1861 actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acctggagac
     1921 tacaaggacg acgacgataa aggagattac aaagatgacg atgataaggg ccaggattat
     1981 aaggacgacg atgacaaggg cggcgtgtcg aaaggcgagg agctgttcac aggcgtggtc
     2041 cccatcctgg ttgagctgga cggtgacgtt aacggccaca aatttagcgt acgtggtgag
     2101 ggcgagggtg acgctaccaa cggaaagctg accctgaaat tcatctgcac aactggcaag
     2161 ctgccggtgc cctggcccac gctggtaacc actctgggcg gaggcgttca atgtttcagt
     2221 cgttacccgg atcacgtgaa gcagcatgac ttcttcaagt cggccgtgcc cgaaggctac
     2281 gtgcaggagc gcaccatcag ttttaaggat gacggcacct acaagactcg cgccgaggtc
     2341 aagttcgaag gagacaccct ggttaatcgt atcgagctca aaggcatcga tttcaaggag
     2401 gacggaaata ttctgggcca caagctggag tacaatttta acagccacaa tgtttacatt
     2461 actgccgaca agcagaagaa cggcatcaag gctaatttca agattcgtca caatgtggag
     2521 ggaggcgact ataaggatga cgatgacaaa caacaagact ataaggacga tgatgacaaa
     2581 ggcggccgcc aagattacaa ggacgatgac gataagcagg gccaagatta caaggatgat
     2641 gatgataagg gaggcgacgg aagcgtgcag ctggcagatc attaccaaca gaacacgccc
     2701 attggtgacg gtccagtcct gctgcctgat aaccactact tgtccaccca gtcggtgctg
     2761 tcgaaggacc cgaacgagaa gcgtgaccac gtggtgctgc tggagttcgt gaccgccgca
     2821 ggaatcaccc tcggcgtgga tgagctttac aagggcggag attataaaga tgacgacgac
     2881 aagcaaggcg actacaaaga tgatgatgac aagggtgact acaaagacga cgatgataaa
     2941 ggagttactg gagaaggaag gggcagtctt ctaacatgcg gtgacgtgga ggagaatccc
     3001 ggccctttaa ttaactctag actggacaag agcaaagtca taaactctgc tctggaatta
     3061 ctcaatgaag tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa gctgggagtt
     3121 gagcagccta ccctgtactg gcacgtgaag aacaagcggg ccctgctcga tgccctggca
     3181 atcgagatgc tggacaggca tcatacccac ttctgccccc tggaaggcga gtcatggcaa
     3241 gactttctgc ggaacaacgc caagtcattc cgctgtgctc tcctctcaca tcgcgacggg
     3301 gctaaagtgc atctcggcac ccgcccaaca gagaaacagt acgaaaccct ggaaaatcag
     3361 ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg cactgtacgc tctgtccgcc
     3421 gtgggccact ttacactggg ctgcgtattg gaggatcagg agcatcaagt agcaaaagag
     3481 gaaagagaga cacctaccac cgattctatg cccccacttc tgagacaagc aattgagctg
     3541 ttcgaccatc agggagccga acctgccttc cttttcggcc tggaactaat catatgtggc
     3601 ctggaaaaac agctaaagtg cgaaagcggc ggaccggccg acgcccttga cgattttgac
     3661 ttagacatgc tcccagccga tgcccttgac gactttgacc ttgatatgct gcctgctgac
     3721 gctcttgacg attttgacct tgacatgctc cccgggtaag ctagcaattc cgctcgagat
     3781 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     3841 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     3901 atggctttca ttttctcctc cttgtataaa tcctggttag ttcttgccac ggcggaactc
     3961 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     4021 gtggtgttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat ctagctttat
     4081 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     4141 taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt gggaggtttt
     4201 ttaaagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4261 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4321 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     4381 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     4441 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4501 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4561 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4621 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4681 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4741 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     4801 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4861 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     4921 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4981 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5041 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     5101 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     5161 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5221 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5281 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5341 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5401 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5461 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5521 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5581 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5641 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5701 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5761 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5821 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5881 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     5941 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6001 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6061 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6121 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6181 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6241 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6301 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6361 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6421 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6481 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6541 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6601 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6661 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6721 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aaca
//
