LOCUS       sequence-383912-        6517 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mCherry, sesRNA in mammalian cells, with GFP as 
            efRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6517)
  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 6517)
  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: CAG-mCherry-sesRNA-GFP-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..6517
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     131..150
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(369..473)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(499..954)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(636..657)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     848..867
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   1026..1155
                     /label=AAV2 ITR
     primer_bind     2021..2043
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2105..2124
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2157..2166
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2163..2870
                     /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(2308..2326)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2576..2595
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2871..2927
                     /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             3195..3248
                     /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(3258..3260,3261..3974)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3258..3260,3261..3971)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3300..3321)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3561..3580)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3908..3929
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3986..4574
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4039..4059)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4457..4468)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4617..4738
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4628..4647)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4682..4701
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4900..5029
                     /label=AAV2 ITR
     rep_origin      complement(5266..5854)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5346..5365)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6025..6517,1..299,300..368))
                     /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"
ORIGIN
        1 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
       61 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
      121 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
      181 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      241 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      301 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      361 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      421 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      481 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
      541 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
      601 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
      661 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
      721 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
      781 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
      841 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
      901 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
      961 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     1021 gccagctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga
     1081 cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca
     1141 tcactagggg ttccttgtag ttaatgatta acccgccatg ctacttatct acgtagccat
     1201 gctctaggaa gagtaccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     1261 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     1321 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     1381 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     1441 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
     1501 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
     1561 gcgatggggg cggggggggg ggggggsgcg cgccrggggg gggggggggg gggggggggg
     1621 gggggggggs grggsggrgr ggkgcggcgg cagccaatca gagcggcgcg ctccgaaagt
     1681 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
     1741 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
     1801 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
     1861 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
     1921 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggctgtccg
     1981 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     2041 accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     2101 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt ggatccgcca
     2161 ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
     2221 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     2281 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     2341 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
     2401 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
     2461 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
     2521 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     2581 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     2641 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     2701 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
     2761 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     2821 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag gctactaact
     2881 tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacctgaa ttcggtggtt
     2941 ttgcggatat ctccgtgaat caatatgtgc tcgtggatca ggacgcgcag gtagtgtccc
     3001 aacaaccccc aacaattttt actcatcaga tcaataatcg tgtgacctac ggtggagcca
     3061 tagacgctca ggacgccaaa accttcgggc cccaaaatga agagaattgg gaacataccc
     3121 cagctcacga aaaacagcca tgccatgccg gtcacgacct ggcggcaccg accctttggc
     3181 acagtggcgc gccagagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc
     3241 ccggcccttt aattaacgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     3301 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     3361 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     3421 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     3481 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     3541 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     3601 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     3661 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     3721 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     3781 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     3841 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     3901 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     3961 agctgtacaa gtaaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     4021 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     4081 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     4141 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     4201 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     4261 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     4321 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     4381 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     4441 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     4501 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     4561 ggccgcctcc ccgcatcgat accgtcgacc cgggcggccg cttcgagcag acatgataag
     4621 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     4681 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     4741 caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggtttttta
     4801 aagcaagtaa aacctctaca aatgtggtaa aatcgataag gatcttccta gagcatggct
     4861 acgtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt
     4921 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     4981 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcattaatg
     5041 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5101 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5161 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5221 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5281 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5341 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5401 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5461 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5521 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5581 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5641 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5701 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5761 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5821 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5881 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5941 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6001 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6061 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6121 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6181 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6241 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6301 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6361 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6421 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6481 taagttggcc gcagtgttat cactcatggt tatggca
//
