LOCUS       sequence-381709-        6752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mCherry, sesRNA in mammalian cells, with smFlag and 
            tTA2 as efRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6752)
  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 6752)
  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-sesRNA-smFlag-tTA2-W3SL
FEATURES             Location/Qualifiers
     source          1..6752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   70..199
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        244..691
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      711..720
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             717..1424
                     /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(862..880)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1130..1149
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1728..1784
                     /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             1788..1811
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1815..1838
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1845..1868
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2397..2420
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2427..2450
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2463..2486
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2496..2519
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2730..2753
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2760..2783
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2787..2810
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2823..2876
                     /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(2886..3500,3501..3506,3507..3542,3543..3545,3546..3581,
                     3582..3584,3585..3620,3621..3629)
                     /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(3704..3724)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4084..4224
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4084..4213
                     /label=AAV2 ITR
     rep_origin      4299..4754
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4386..4405)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4596..4617
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4771..4790)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4890..4912
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4950..4968)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5036..5140
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5141..5209,5210..6001)
                     /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(5359..5378)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6172..6752,1..8)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6661..6680
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
       61 ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg
      121 gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac
      181 tccatcacta ggggttcctg cggccgcacg cgtgtgtcta gactgcagag ggccctgcgt
      241 atgagtgcaa gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac
      301 cgaccccgac ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc
      361 agagaggggg aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc
      421 gcggacagtg ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg
      481 cgcgctgacg tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt
      541 ccgcgccgcc gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg
      601 gcgcgaccat ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag
      661 cggaggagtc gtgtcgtgcc tgagagcgca gtcgagaagg taccggatcc gccaccatgg
      721 tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
      781 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
      841 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg
      901 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
      961 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     1021 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     1081 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     1141 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     1201 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg
     1261 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     1321 tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg
     1381 ccgagggccg ccactccacc ggcggcatgg acgagctgta caaggaattc acattaagaa
     1441 tcggtctgtt cccaggagtt ctgtctccaa ggtcacacca tgtctgggaa ggaaaactgt
     1501 tccttcacgc aaatgctcgc agcaacttcc cagtaccctt tatccttgtg tagccacctg
     1561 cctcgctctc tacactgcag agactgatga agcactgtga gccagggcca tggagcagga
     1621 gaagggctgc tttcacggaa ccacagtcag caagacatcg gggattacgt gatttccagc
     1681 gagggttcaa gtatcgagtc cataagaaag gtgagtccgg cgcgccagct actaacttca
     1741 gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acctggagac tacaaggacg
     1801 acgacgataa aggagattac aaagatgacg atgataaggg ccaggattat aaggacgacg
     1861 atgacaaggg cggcgtgtcg aaaggcgagg agctgttcac aggcgtggtc cccatcctgg
     1921 ttgagctgga cggtgacgtt aacggccaca aatttagcgt acgtggtgag ggcgagggtg
     1981 acgctaccaa cggaaagctg accctgaaat tcatctgcac aactggcaag ctgccggtgc
     2041 cctggcccac gctggtaacc actctgggcg gaggcgttca atgtttcagt cgttacccgg
     2101 atcacgtgaa gcagcatgac ttcttcaagt cggccgtgcc cgaaggctac gtgcaggagc
     2161 gcaccatcag ttttaaggat gacggcacct acaagactcg cgccgaggtc aagttcgaag
     2221 gagacaccct ggttaatcgt atcgagctca aaggcatcga tttcaaggag gacggaaata
     2281 ttctgggcca caagctggag tacaatttta acagccacaa tgtttacatt actgccgaca
     2341 agcagaagaa cggcatcaag gctaatttca agattcgtca caatgtggag ggaggcgact
     2401 ataaggatga cgatgacaaa caacaagact ataaggacga tgatgacaaa ggcggccgcc
     2461 aagattacaa ggacgatgac gataagcagg gccaagatta caaggatgat gatgataagg
     2521 gaggcgacgg aagcgtgcag ctggcagatc attaccaaca gaacacgccc attggtgacg
     2581 gtccagtcct gctgcctgat aaccactact tgtccaccca gtcggtgctg tcgaaggacc
     2641 cgaacgagaa gcgtgaccac gtggtgctgc tggagttcgt gaccgccgca ggaatcaccc
     2701 tcggcgtgga tgagctttac aagggcggag attataaaga tgacgacgac aagcaaggcg
     2761 actacaaaga tgatgatgac aagggtgact acaaagacga cgatgataaa ggagttactg
     2821 gagaaggaag gggcagtctt ctaacatgcg gtgacgtgga ggagaatccc ggccctttaa
     2881 ttaactctag actggacaag agcaaagtca taaactctgc tctggaatta ctcaatgaag
     2941 tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa gctgggagtt gagcagccta
     3001 ccctgtactg gcacgtgaag aacaagcggg ccctgctcga tgccctggca atcgagatgc
     3061 tggacaggca tcatacccac ttctgccccc tggaaggcga gtcatggcaa gactttctgc
     3121 ggaacaacgc caagtcattc cgctgtgctc tcctctcaca tcgcgacggg gctaaagtgc
     3181 atctcggcac ccgcccaaca gagaaacagt acgaaaccct ggaaaatcag ctcgcgttcc
     3241 tgtgtcagca aggcttctcc ctggagaacg cactgtacgc tctgtccgcc gtgggccact
     3301 ttacactggg ctgcgtattg gaggatcagg agcatcaagt agcaaaagag gaaagagaga
     3361 cacctaccac cgattctatg cccccacttc tgagacaagc aattgagctg ttcgaccatc
     3421 agggagccga acctgccttc cttttcggcc tggaactaat catatgtggc ctggaaaaac
     3481 agctaaagtg cgaaagcggc ggaccggccg acgcccttga cgattttgac ttagacatgc
     3541 tcccagccga tgcccttgac gactttgacc ttgatatgct gcctgctgac gctcttgacg
     3601 attttgacct tgacatgctc cccgggtaag ctagcaattc cgctcgagat aatcaacctc
     3661 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     3721 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     3781 ttttctcctc cttgtataaa tcctggttag ttcttgccac ggcggaactc atcgccgcct
     3841 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttta
     3901 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat ctagctttat ttgtgaaatt
     3961 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     4021 aattgcattc attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcggc
     4081 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     4141 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     4201 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     4261 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     4321 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     4381 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4441 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4501 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4561 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4621 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     4681 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     4741 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4801 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4861 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4921 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     4981 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5041 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5101 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5161 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5221 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5281 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5341 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5401 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5461 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5521 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5581 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5641 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5701 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     5761 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5821 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     5881 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5941 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6001 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6061 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6121 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6181 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6241 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6301 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6361 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6421 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6481 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6541 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6601 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6661 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6721 atttttgtga tgctcgtcag gggggcggag cc
//
