LOCUS       sequence-481740-        6758 bp    DNA     circular SYN 01-AUG-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 6758)
  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 6758)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 1, 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-Plxnd1
            sesRNA-2a-msFlag-2a-t...
FEATURES             Location/Qualifiers
     source          1..6758
                     /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             1852..1908
                     /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             1912..1935
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1939..1962
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1969..1992
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2521..2544
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2551..2574
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2587..2610
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2620..2643
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2854..2877
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2884..2907
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2911..2934
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2947..3000
                     /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(3010..3624,3625..3630,3631..3666,3667..3669,3670..3705,
                     3706..3708,3709..3744,3745..3753)
                     /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(3828..3848)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4208..4348
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4208..4337
                     /label=AAV2 ITR
     rep_origin      4423..4878
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4510..4529)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4720..4741
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4895..4914)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5014..5036
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5074..5092)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5160..5264
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5265..5333,5334..6125)
                     /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(5483..5502)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6296..6758,1..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 aggaattcgc ggggcaggca
     1561 gatgcgcgcc agcaggctgc gcgcctggct gtccttgtcg cccgcacgcg cctcgctgtt
     1621 gagcgccagg tacgcgtagg gctgcgcgct gtgcggtagc accgccgggt gcaagaaggc
     1681 acgcacgaag cccagcttgt gctgctcctt ggcgccctgc ttgatcttca ggatgttatc
     1741 gtccgacggg ttaaggtcga aggtgaagag cttggccaag tctccacgcg cgtccaggga
     1801 gcggatagcg atctcgggcg tgttctcgaa gcggtggtct tcggcgcgcc agctactaac
     1861 ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctgg agactacaag
     1921 gacgacgacg ataaaggaga ttacaaagat gacgatgata agggccagga ttataaggac
     1981 gacgatgaca agggcggcgt gtcgaaaggc gaggagctgt tcacaggcgt ggtccccatc
     2041 ctggttgagc tggacggtga cgttaacggc cacaaattta gcgtacgtgg tgagggcgag
     2101 ggtgacgcta ccaacggaaa gctgaccctg aaattcatct gcacaactgg caagctgccg
     2161 gtgccctggc ccacgctggt aaccactctg ggcggaggcg ttcaatgttt cagtcgttac
     2221 ccggatcacg tgaagcagca tgacttcttc aagtcggccg tgcccgaagg ctacgtgcag
     2281 gagcgcacca tcagttttaa ggatgacggc acctacaaga ctcgcgccga ggtcaagttc
     2341 gaaggagaca ccctggttaa tcgtatcgag ctcaaaggca tcgatttcaa ggaggacgga
     2401 aatattctgg gccacaagct ggagtacaat tttaacagcc acaatgttta cattactgcc
     2461 gacaagcaga agaacggcat caaggctaat ttcaagattc gtcacaatgt ggagggaggc
     2521 gactataagg atgacgatga caaacaacaa gactataagg acgatgatga caaaggcggc
     2581 cgccaagatt acaaggacga tgacgataag cagggccaag attacaagga tgatgatgat
     2641 aagggaggcg acggaagcgt gcagctggca gatcattacc aacagaacac gcccattggt
     2701 gacggtccag tcctgctgcc tgataaccac tacttgtcca cccagtcggt gctgtcgaag
     2761 gacccgaacg agaagcgtga ccacgtggtg ctgctggagt tcgtgaccgc cgcaggaatc
     2821 accctcggcg tggatgagct ttacaagggc ggagattata aagatgacga cgacaagcaa
     2881 ggcgactaca aagatgatga tgacaagggt gactacaaag acgacgatga taaaggagtt
     2941 actggagaag gaaggggcag tcttctaaca tgcggtgacg tggaggagaa tcccggccct
     3001 ttaattaact ctagactgga caagagcaaa gtcataaact ctgctctgga attactcaat
     3061 gaagtcggta tcgaaggcct gacgacaagg aaactcgctc aaaagctggg agttgagcag
     3121 cctaccctgt actggcacgt gaagaacaag cgggccctgc tcgatgccct ggcaatcgag
     3181 atgctggaca ggcatcatac ccacttctgc cccctggaag gcgagtcatg gcaagacttt
     3241 ctgcggaaca acgccaagtc attccgctgt gctctcctct cacatcgcga cggggctaaa
     3301 gtgcatctcg gcacccgccc aacagagaaa cagtacgaaa ccctggaaaa tcagctcgcg
     3361 ttcctgtgtc agcaaggctt ctccctggag aacgcactgt acgctctgtc cgccgtgggc
     3421 cactttacac tgggctgcgt attggaggat caggagcatc aagtagcaaa agaggaaaga
     3481 gagacaccta ccaccgattc tatgccccca cttctgagac aagcaattga gctgttcgac
     3541 catcagggag ccgaacctgc cttccttttc ggcctggaac taatcatatg tggcctggaa
     3601 aaacagctaa agtgcgaaag cggcggaccg gccgacgccc ttgacgattt tgacttagac
     3661 atgctcccag ccgatgccct tgacgacttt gaccttgata tgctgcctgc tgacgctctt
     3721 gacgattttg accttgacat gctccccggg taagctagca attccgctcg agataatcaa
     3781 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     3841 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     3901 ttcattttct cctccttgta taaatcctgg ttagttcttg ccacggcgga actcatcgcc
     3961 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     4021 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccatctagct ttatttgtga
     4081 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     4141 caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag
     4201 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     4261 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     4321 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     4381 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     4441 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     4501 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4561 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4621 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4681 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     4741 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     4801 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     4861 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4921 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4981 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     5041 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     5101 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     5161 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5221 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5281 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5341 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5401 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5461 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5521 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5581 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5641 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5701 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5761 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5821 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5881 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5941 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     6001 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6061 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6121 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6181 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6241 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6301 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6361 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6421 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6481 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6541 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6601 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6661 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6721 aggcggacag gtatccggta agcggcaggg tcggaaca
//
