LOCUS       sequence-487716-        6459 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Expression of BFP, sesRNA in mammalian cells, with GFP as efRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6459)
  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 6459)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 2, 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-BFP-sesRNA-GFP-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..6459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(249..353)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(379..834)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(516..537)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     728..747
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   906..1035
                     /label=AAV2 ITR
     promoter        1346..1623
                     /label=chicken beta-actin promoter
     primer_bind     1902..1924
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1986..2005
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2044..2742
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNYMALK
                     LVGGSHLIANAKTTYRSKKPANNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             2749..2805
                     /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"
     primer_bind     complement(2923..2942)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3016..3069
                     /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(3079..3081,3082..3795)
                     /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(3079..3081,3082..3792)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3121..3142)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3382..3401)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3729..3750
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3808..4396
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3861..3881)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4279..4290)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4439..4560
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4450..4469)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4504..4523
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4722..4851
                     /label=AAV2 ITR
     rep_origin      complement(5088..5676)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5168..5187)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5847..6459,1..179,180..248))
                     /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 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
       61 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      121 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      181 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      241 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      301 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      361 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
      421 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
      481 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
      541 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
      601 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
      661 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
      721 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
      781 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
      841 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
      901 gccagctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga
      961 cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca
     1021 tcactagggg ttccttgtag ttaatgatta acccgccatg ctacttatct acgtagccat
     1081 gctctaggaa gagtaccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     1141 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     1201 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     1261 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     1321 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
     1381 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
     1441 gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg
     1501 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
     1561 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
     1621 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
     1681 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
     1741 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
     1801 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggctgtcc
     1861 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1921 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1981 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tggatccgcc
     2041 accatgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg
     2101 gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag
     2161 accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct
     2221 actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc
     2281 ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg
     2341 ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc
     2401 aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc
     2461 tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg cagaaactac
     2521 atggccctga agctcgtggg cgggagccat ctgatcgcaa acgccaagac cacatataga
     2581 tccaagaaac ccgctaataa cctcaagatg cctggcgtct actatgtgga ctacagactg
     2641 gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt ggcagtggcc
     2701 agatactgcg acctccctag caaactgggg cacaagctca atactagtgc tactaacttc
     2761 agcctgctga agcaggctgg agacgtggag gagaaccctg gacctgaatt cactccacca
     2821 ggtagtggcc gccgtccttc agcttcaggg cctggtggat ctcgcccttc agcacgccgt
     2881 cgcgggggta caggcgctcg gtggaggcct cccagcccat agtcttcttc tgcattacgg
     2941 ggccgtcggg ggggaagttg gtgccgcgca tcttcacctt gtagatcagc gtgccgtcct
     3001 gcagggggcg cgccagaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat
     3061 cccggccctt taattaacgt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     3121 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     3181 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     3241 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     3301 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     3361 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     3421 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     3481 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     3541 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     3601 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     3661 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     3721 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     3781 gagctgtaca agtaaaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga
     3841 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     3901 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     3961 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     4021 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     4081 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     4141 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     4201 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     4261 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     4321 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     4381 tgggccgcct ccccgcatcg ataccgtcga cccgggcggc cgcttcgagc agacatgata
     4441 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     4501 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     4561 aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg ggaggttttt
     4621 taaagcaagt aaaacctcta caaatgtggt aaaatcgata aggatcttcc tagagcatgg
     4681 ctacgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc tagtgatgga
     4741 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     4801 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcattaa
     4861 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4921 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4981 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5041 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5101 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5161 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5221 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5281 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5341 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5401 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5461 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5521 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5581 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5641 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5701 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5761 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5821 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5881 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5941 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6001 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6061 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6121 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6181 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6241 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6301 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6361 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6421 gaatagtgta tgcggcgacc gagttgctct tgcccggcg
//
