LOCUS       sequence-469474-        6857 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 6857)
  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 6857)
  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-Rorb sesRNA-2a-msFlag-2a-tTA...
FEATURES             Location/Qualifiers
     source          1..6857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      169..757
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     658..677
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   819..948
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        993..1440
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      1460..1469
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1466..2173
                     /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(1611..1629)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1879..1898
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2582..2638
                     /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             2642..2665
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2669..2692
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2699..2722
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3251..3274
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3281..3304
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3317..3340
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3350..3373
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3584..3607
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3614..3637
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3641..3664
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3677..3730
                     /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(3740..4354,4355..4360,4361..4396,4397..4399,4400..4435,
                     4436..4438,4439..4474,4475..4483)
                     /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(4558..4578)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4938..5078
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4938..5067
                     /label=AAV2 ITR
     rep_origin      5153..5608
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5240..5259)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5450..5471
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5625..5644)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5744..5766
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5804..5822)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5890..5994
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5995..6063,6064..6855)
                     /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(6213..6232)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
       61 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
      121 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
      181 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
      241 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
      301 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
      361 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
      421 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
      481 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
      541 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
      601 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      661 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      721 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      781 acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc tgcgcgctcg
      841 ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
      901 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc ggccgcacgc
      961 gtgtgtctag actgcagagg gccctgcgta tgagtgcaag tgggttttag gaccaggatg
     1021 aggcggggtg ggggtgccta cctgacgacc gaccccgacc cactggacaa gcacccaacc
     1081 cccattcccc aaattgcgca tcccctatca gagaggggga ggggaaacag gatgcggcga
     1141 ggcgcgtgcg cactgccagc ttcagcaccg cggacagtgc cttcgccccc gcctggcggc
     1201 gcgcgccacc gccgcctcag cactgaaggc gcgctgacgt cactcgccgg tcccccgcaa
     1261 actccccttc ccggccacct tggtcgcgtc cgcgccgccg ccggcccagc cggaccgcac
     1321 cacgcgaggc gcgagatagg ggggcacggg cgcgaccatc tgcgctgcgg cgccggcgac
     1381 tcagcgctgc ctcagtctgc ggtgggcagc ggaggagtcg tgtcgtgcct gagagcgcag
     1441 tcgagaaggt accggatccg ccaccatggt gagcaagggc gaggaggata acatggccat
     1501 catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt
     1561 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     1621 ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta
     1681 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     1741 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     1801 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     1861 caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc
     1921 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct
     1981 gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt
     2041 gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga
     2101 ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga
     2161 cgagctgtac aaggaattct acgatctcag acattgttat cccgggagcg aactgcccgt
     2221 tgttgaaaga gctataggta aacaagttgg gtacagatgt gaggtcatag ataggttctt
     2281 gctttatctg tttgatccca gtcatgtcca gctcctgact gatcgggaga cggctgaccg
     2341 gaatctatgc tgtaataacc ttcggactta ggcaggtcaa tgcgtgcccg ttggcgtatg
     2401 tgccgccggt ctcggtgttc aggttgctga ggccattgct aatgctgctg ctgtacaccc
     2461 tggcgagggc ctccgcctcc ccactctgct gctgccgctg ctcctgcagc ctttgctgag
     2521 gcttctgcac ctcagcatac aggctgtccc gctgcttctt ggacatcctc ccggcgcgcc
     2581 agctactaac ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctgg
     2641 agactacaag gacgacgacg ataaaggaga ttacaaagat gacgatgata agggccagga
     2701 ttataaggac gacgatgaca agggcggcgt gtcgaaaggc gaggagctgt tcacaggcgt
     2761 ggtccccatc ctggttgagc tggacggtga cgttaacggc cacaaattta gcgtacgtgg
     2821 tgagggcgag ggtgacgcta ccaacggaaa gctgaccctg aaattcatct gcacaactgg
     2881 caagctgccg gtgccctggc ccacgctggt aaccactctg ggcggaggcg ttcaatgttt
     2941 cagtcgttac ccggatcacg tgaagcagca tgacttcttc aagtcggccg tgcccgaagg
     3001 ctacgtgcag gagcgcacca tcagttttaa ggatgacggc acctacaaga ctcgcgccga
     3061 ggtcaagttc gaaggagaca ccctggttaa tcgtatcgag ctcaaaggca tcgatttcaa
     3121 ggaggacgga aatattctgg gccacaagct ggagtacaat tttaacagcc acaatgttta
     3181 cattactgcc gacaagcaga agaacggcat caaggctaat ttcaagattc gtcacaatgt
     3241 ggagggaggc gactataagg atgacgatga caaacaacaa gactataagg acgatgatga
     3301 caaaggcggc cgccaagatt acaaggacga tgacgataag cagggccaag attacaagga
     3361 tgatgatgat aagggaggcg acggaagcgt gcagctggca gatcattacc aacagaacac
     3421 gcccattggt gacggtccag tcctgctgcc tgataaccac tacttgtcca cccagtcggt
     3481 gctgtcgaag gacccgaacg agaagcgtga ccacgtggtg ctgctggagt tcgtgaccgc
     3541 cgcaggaatc accctcggcg tggatgagct ttacaagggc ggagattata aagatgacga
     3601 cgacaagcaa ggcgactaca aagatgatga tgacaagggt gactacaaag acgacgatga
     3661 taaaggagtt actggagaag gaaggggcag tcttctaaca tgcggtgacg tggaggagaa
     3721 tcccggccct ttaattaact ctagactgga caagagcaaa gtcataaact ctgctctgga
     3781 attactcaat gaagtcggta tcgaaggcct gacgacaagg aaactcgctc aaaagctggg
     3841 agttgagcag cctaccctgt actggcacgt gaagaacaag cgggccctgc tcgatgccct
     3901 ggcaatcgag atgctggaca ggcatcatac ccacttctgc cccctggaag gcgagtcatg
     3961 gcaagacttt ctgcggaaca acgccaagtc attccgctgt gctctcctct cacatcgcga
     4021 cggggctaaa gtgcatctcg gcacccgccc aacagagaaa cagtacgaaa ccctggaaaa
     4081 tcagctcgcg ttcctgtgtc agcaaggctt ctccctggag aacgcactgt acgctctgtc
     4141 cgccgtgggc cactttacac tgggctgcgt attggaggat caggagcatc aagtagcaaa
     4201 agaggaaaga gagacaccta ccaccgattc tatgccccca cttctgagac aagcaattga
     4261 gctgttcgac catcagggag ccgaacctgc cttccttttc ggcctggaac taatcatatg
     4321 tggcctggaa aaacagctaa agtgcgaaag cggcggaccg gccgacgccc ttgacgattt
     4381 tgacttagac atgctcccag ccgatgccct tgacgacttt gaccttgata tgctgcctgc
     4441 tgacgctctt gacgattttg accttgacat gctccccggg taagctagca attccgctcg
     4501 agataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     4561 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     4621 ccgtatggct ttcattttct cctccttgta taaatcctgg ttagttcttg ccacggcgga
     4681 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     4741 ttccgtggtg tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccatctagct
     4801 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     4861 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg
     4921 ttttttaaag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4981 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     5041 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     5101 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     5161 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5221 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5281 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5341 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     5401 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5461 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     5521 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5581 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     5641 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     5701 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     5761 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     5821 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5881 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5941 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     6001 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6061 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6121 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6181 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6241 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6301 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6361 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6421 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6481 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6541 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6601 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6661 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6721 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6781 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6841 attaagcatt ggtaact
//
