LOCUS       sequence-383797-        6490 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression of BFP, sesRNA in mammalian cells, with tTA2 as efRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6490)
  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 6490)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-tTA2-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..6490
                     /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
     primer_bind     1900..1922
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1984..2003
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2042..2740
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNYMALK
                     LVGGSHLIANAKTTYRSKKPANNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             2747..2803
                     /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(2921..2940)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3014..3067
                     /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(3077..3691,3692..3697,3698..3733,3734..3736,3737..3772,
                     3773..3775,3776..3811,3812..3820)
                     /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"
     misc_feature    3839..4427
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3892..3912)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4310..4321)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4470..4591
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4481..4500)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4535..4554
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4753..4882
                     /label=AAV2 ITR
     rep_origin      complement(5119..5707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5199..5218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5878..6490,1..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 gggggsgcgc gccrggsggg gggggggggg gsgggggggg
     1501 gggsggggsg rggcggrgrg gkgcggcggc agccaatcag agcggcgcgc tccgaaagtt
     1561 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
     1621 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
     1681 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
     1741 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
     1801 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggctgtccgc
     1861 ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga
     1921 ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc
     1981 tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg gatccgccac
     2041 catgagcgag ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga
     2101 caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac
     2161 catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac
     2221 tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt
     2281 caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg
     2341 cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa
     2401 gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg
     2461 ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaactacat
     2521 ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac gccaagacca catatagatc
     2581 caagaaaccc gctaataacc tcaagatgcc tggcgtctac tatgtggact acagactgga
     2641 aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag
     2701 atactgcgac ctccctagca aactggggca caagctcaat actagtgcta ctaacttcag
     2761 cctgctgaag caggctggag acgtggagga gaaccctgga cctgaattca ctccaccagg
     2821 tagtggccgc cgtccttcag cttcagggcc tggtggatct cgcccttcag cacgccgtcg
     2881 cgggggtaca ggcgctcggt ggaggcctcc cagcccatag tcttcttctg cattacgggg
     2941 ccgtcggggg ggaagttggt gccgcgcatc ttcaccttgt agatcagcgt gccgtcctgc
     3001 agggggcgcg ccagagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc
     3061 cggcccttta attaactcta gactggacaa gagcaaagtc ataaactctg ctctggaatt
     3121 actcaatgaa gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt
     3181 tgagcagcct accctgtact ggcacgtgaa gaacaagcgg gccctgctcg atgccctggc
     3241 aatcgagatg ctggacaggc atcataccca cttctgcccc ctggaaggcg agtcatggca
     3301 agactttctg cggaacaacg ccaagtcatt ccgctgtgct ctcctctcac atcgcgacgg
     3361 ggctaaagtg catctcggca cccgcccaac agagaaacag tacgaaaccc tggaaaatca
     3421 gctcgcgttc ctgtgtcagc aaggcttctc cctggagaac gcactgtacg ctctgtccgc
     3481 cgtgggccac tttacactgg gctgcgtatt ggaggatcag gagcatcaag tagcaaaaga
     3541 ggaaagagag acacctacca ccgattctat gcccccactt ctgagacaag caattgagct
     3601 gttcgaccat cagggagccg aacctgcctt ccttttcggc ctggaactaa tcatatgtgg
     3661 cctggaaaaa cagctaaagt gcgaaagcgg cggaccggcc gacgcccttg acgattttga
     3721 cttagacatg ctcccagccg atgcccttga cgactttgac cttgatatgc tgcctgctga
     3781 cgctcttgac gattttgacc ttgacatgct ccccgggtaa gctagcaagc ttatcgataa
     3841 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3901 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3961 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     4021 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     4081 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     4141 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     4201 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     4261 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     4321 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     4381 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg
     4441 acccgggcgg ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac
     4501 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4561 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     4621 tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     4681 taaaatcgat aaggatcttc ctagagcatg gctacgtaga taagtagcat ggcgggttaa
     4741 tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     4801 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     4861 cagtgagcga gcgagcgcgc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4921 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4981 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5041 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5101 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5161 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5221 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5281 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5341 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5401 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5461 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5521 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5581 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5641 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5701 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5761 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5821 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5881 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5941 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6001 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6061 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6121 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6181 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6241 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6301 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6361 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6421 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6481 ttgcccggcg
//
