LOCUS       sequence-472759-        7172 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 7172)
  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 7172)
  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-Satb2 sesRNA-2a-msFlag-2a-tT...
FEATURES             Location/Qualifiers
     source          1..7172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      93..681
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     582..601
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   743..872
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        917..1364
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      1384..1393
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1390..2097
                     /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(1535..1553)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1803..1822
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2821..2877
                     /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             2881..2904
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2908..2931
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2938..2961
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3490..3513
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3520..3543
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3556..3579
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3589..3612
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3823..3846
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3853..3876
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3880..3903
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3916..3969
                     /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(3979..4593,4594..4599,4600..4635,4636..4638,4639..4674,
                     4675..4677,4678..4713,4714..4722)
                     /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(4797..4817)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   5177..5317
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5177..5306
                     /label=AAV2 ITR
     rep_origin      5392..5847
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5479..5498)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5689..5710
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5864..5883)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5983..6005
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6043..6061)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6129..6233
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6234..6302,6303..7094)
                     /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(6452..6471)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
       61 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
      121 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
      181 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
      241 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
      301 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
      361 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
      421 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
      481 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
      541 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
      601 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
      661 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
      721 tgctggcctt ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc
      781 gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga
      841 gggagtggcc aactccatca ctaggggttc ctgcggccgc acgcgtgtgt ctagactgca
      901 gagggccctg cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg
      961 cctacctgac gaccgacccc gacccactgg acaagcaccc aacccccatt ccccaaattg
     1021 cgcatcccct atcagagagg gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc
     1081 cagcttcagc accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc
     1141 tcagcactga aggcgcgctg acgtcactcg ccggtccccc gcaaactccc cttcccggcc
     1201 accttggtcg cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg aggcgcgaga
     1261 taggggggca cgggcgcgac catctgcgct gcggcgccgg cgactcagcg ctgcctcagt
     1321 ctgcggtggg cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaga aggtaccgga
     1381 tccgccacca tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
     1441 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
     1501 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     1561 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
     1621 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
     1681 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     1741 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     1801 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
     1861 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     1921 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     1981 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     2041 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaaggaa
     2101 ttcttatctc tggtcggttt cggccggtgc agccttgctc ttgtcggcat tctcttcctc
     2161 ggcctccacc ttgtacatct cctccgagcc ttcctcactg tcattctcct cggactcggt
     2221 cagcagctcc tcgtccttat actctgccac gtccacggcg gagcccaggt gctccttcag
     2281 cttgccatgg tgcttcacgt ggtacctctg gttctggaag aacttgatga tggtgtgttt
     2341 ggggagatcc agctgggcgg agagtgtgtg gatggcttcc tggtcgggat agaggcctac
     2401 atcatggatg aagctttgaa ggatgcccag ggcttccaaa gagatctttg tgcgagaccg
     2461 aggctttttg gcacagctgt cttctgttag aggaggcggt aggggtgctt cttctctggg
     2521 aggggagctc tccttagttg gctgggactg ctgtcgatga aggacctgca cgggctcagg
     2581 tgggagctgg accacaggct gcatgcgttc actgtggtga ggtcgagatt cttcctcata
     2641 gatcacatcc cgctcaggtt ggggaagatt caggaaacgg cggatggtgc agagattctc
     2701 ccaaagggtg cggttttctg ggctggggtt ctccttccaa cgaagcagtt cgcaaagcca
     2761 gccctgactt ttgtttgcag ccactttggc aaacagggct tgagacacct tggcgcgcca
     2821 gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacctgga
     2881 gactacaagg acgacgacga taaaggagat tacaaagatg acgatgataa gggccaggat
     2941 tataaggacg acgatgacaa gggcggcgtg tcgaaaggcg aggagctgtt cacaggcgtg
     3001 gtccccatcc tggttgagct ggacggtgac gttaacggcc acaaatttag cgtacgtggt
     3061 gagggcgagg gtgacgctac caacggaaag ctgaccctga aattcatctg cacaactggc
     3121 aagctgccgg tgccctggcc cacgctggta accactctgg gcggaggcgt tcaatgtttc
     3181 agtcgttacc cggatcacgt gaagcagcat gacttcttca agtcggccgt gcccgaaggc
     3241 tacgtgcagg agcgcaccat cagttttaag gatgacggca cctacaagac tcgcgccgag
     3301 gtcaagttcg aaggagacac cctggttaat cgtatcgagc tcaaaggcat cgatttcaag
     3361 gaggacggaa atattctggg ccacaagctg gagtacaatt ttaacagcca caatgtttac
     3421 attactgccg acaagcagaa gaacggcatc aaggctaatt tcaagattcg tcacaatgtg
     3481 gagggaggcg actataagga tgacgatgac aaacaacaag actataagga cgatgatgac
     3541 aaaggcggcc gccaagatta caaggacgat gacgataagc agggccaaga ttacaaggat
     3601 gatgatgata agggaggcga cggaagcgtg cagctggcag atcattacca acagaacacg
     3661 cccattggtg acggtccagt cctgctgcct gataaccact acttgtccac ccagtcggtg
     3721 ctgtcgaagg acccgaacga gaagcgtgac cacgtggtgc tgctggagtt cgtgaccgcc
     3781 gcaggaatca ccctcggcgt ggatgagctt tacaagggcg gagattataa agatgacgac
     3841 gacaagcaag gcgactacaa agatgatgat gacaagggtg actacaaaga cgacgatgat
     3901 aaaggagtta ctggagaagg aaggggcagt cttctaacat gcggtgacgt ggaggagaat
     3961 cccggccctt taattaactc tagactggac aagagcaaag tcataaactc tgctctggaa
     4021 ttactcaatg aagtcggtat cgaaggcctg acgacaagga aactcgctca aaagctggga
     4081 gttgagcagc ctaccctgta ctggcacgtg aagaacaagc gggccctgct cgatgccctg
     4141 gcaatcgaga tgctggacag gcatcatacc cacttctgcc ccctggaagg cgagtcatgg
     4201 caagactttc tgcggaacaa cgccaagtca ttccgctgtg ctctcctctc acatcgcgac
     4261 ggggctaaag tgcatctcgg cacccgccca acagagaaac agtacgaaac cctggaaaat
     4321 cagctcgcgt tcctgtgtca gcaaggcttc tccctggaga acgcactgta cgctctgtcc
     4381 gccgtgggcc actttacact gggctgcgta ttggaggatc aggagcatca agtagcaaaa
     4441 gaggaaagag agacacctac caccgattct atgcccccac ttctgagaca agcaattgag
     4501 ctgttcgacc atcagggagc cgaacctgcc ttccttttcg gcctggaact aatcatatgt
     4561 ggcctggaaa aacagctaaa gtgcgaaagc ggcggaccgg ccgacgccct tgacgatttt
     4621 gacttagaca tgctcccagc cgatgccctt gacgactttg accttgatat gctgcctgct
     4681 gacgctcttg acgattttga ccttgacatg ctccccgggt aagctagcaa ttccgctcga
     4741 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     4801 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     4861 cgtatggctt tcattttctc ctccttgtat aaatcctggt tagttcttgc cacggcggaa
     4921 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     4981 tccgtggtgt ttatttgtga aatttgtgat gctattgctt tatttgtaac catctagctt
     5041 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     5101 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt
     5161 tttttaaagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     5221 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     5281 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     5341 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     5401 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5461 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5521 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5581 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     5641 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5701 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     5761 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5821 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     5881 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     5941 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     6001 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     6061 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     6121 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     6181 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     6241 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     6301 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     6361 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     6421 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     6481 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6541 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6601 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6661 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6721 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6781 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6841 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6901 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6961 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     7021 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     7081 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     7141 ttcattttta atttaaaagg atctaggtga ag
//
