LOCUS       sequence-492549-        5527 bp    DNA     circular SYN 11-NOV-2025
DEFINITION  Expression plasmid for rtTA-2A-tagBFP driven from the CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5527)
  AUTHORS   Kabaria SR, Bae Y, Ehmann ME, Lende-Dorn BA, Beitz AM, Peterman EL, 
            Love KS, Ploessl DS, Galloway KE
  TITLE     Programmable promoter editing for precise control of transgene 
            expression.
  JOURNAL   Nat Biotechnol. 2025 Oct 13. doi: 10.1038/s41587-025-02854-y.
  PUBMED    41083901
REFERENCE   2  (bases 1 to 5527)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 11, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2025 Oct 13. doi: 10.1038/s41587-025-02854-y."
COMMENT     Sequence Label: CAG-rtTA-2A-tagBFP-bGH_pKG3264
FEATURES             Location/Qualifiers
     source          1..5527
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(111..130)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             122..931
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(189..208)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1021..1609
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1510..1529
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      1774..1860
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1952..1979
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     1987..2010
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     enhancer        2109..2412
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          2692..3708
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3632..3654
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3716..3735
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3768..4511
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             4521..4577
                     /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             4590..5291
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    5303..5527
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 cgctcgctga ctgcaggtgc ccgtgtctca aaatctctga tgttacattg cacaagataa
       61 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
      121 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
      181 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
      241 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
      301 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
      361 gaccatcaag cattttatcc gtactcctgg tgatgcatgg ttactcacca ctgcgatccc
      421 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
      481 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
      541 cagcgatcgc gtatttcgtc tagctcaggc gcaatcacga atgaataacg gtttggttga
      601 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
      661 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
      721 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
      781 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
      841 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
      901 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacatta
      961 ttcagattgg gccccgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     1021 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     1081 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     1141 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     1201 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1261 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1321 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     1381 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     1441 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     1501 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     1561 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     1621 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     1681 gttatcccct gattctgtgg ataaccgtat taccgctagc atggatctcg gggacgtcta
     1741 actactaagc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcggaa
     1801 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     1861 ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg ggcaggacgc
     1921 ccgccataaa ctgccaggca tcaaactaag cagaaggcca tcctgacgga tggccttttt
     1981 gcgtttctac aaactcttcc tgttagttag ttacttaagc tcgggcccac ctgcagtcta
     2041 cttactacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     2101 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     2161 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     2221 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     2281 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     2341 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     2401 gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc
     2461 tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg
     2521 gggggggggg gggggggcgc cccggggggg gggggggggg gggggggggg gggggggggg
     2581 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
     2641 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     2701 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
     2761 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
     2821 aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag
     2881 gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg
     2941 tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc
     3001 ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc
     3061 ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg
     3121 gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc
     3181 cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg
     3241 ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct
     3301 cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga
     3361 ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt
     3421 cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc
     3481 gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt
     3541 gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac
     3601 ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc
     3661 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac
     3721 gtgctggtta ttgtgctgtc tcatcatttt ggcaaaaatg tgccaccatg agccggctgg
     3781 acaagagcaa agtgatcaac agcgccctgg aactgctgaa cggcgtgggc atcgagggcc
     3841 tgaccacccg gaagctggcc cagaaactgg gcgtggaaca gcccaccctg tactggcacg
     3901 tgaagaacaa gcgggccctg ctggacgccc tgcccatcga gatgctggac cggcaccaca
     3961 cccacagctg ccctctggaa ggcgagagct ggcaggactt cctgcggaac aacgccaaga
     4021 gctacagatg cgccctgctg agccaccggg acggcgccaa agtgcacctg ggcaccagac
     4081 ccaccgagaa gcagtacgag acactggaaa accagctggc cttcctgtgc cagcagggct
     4141 tcagcctgga aaacgccctg tacgccctga gcgccgtggg ccacttcacc ctgggctgcg
     4201 tgctggaaga acaggaacac caggtcgcca aagaggaaag agagacaccc accaccgaca
     4261 gcatgccccc cctgctgaag caggccatcg agctgttcga cagacagggc gccgagcccg
     4321 ccttcctgtt cggcctggaa ctgatcatct gcggcctcga gaagcagctg aagtgcgaga
     4381 gcggcggacc caccgacgcc ctggacgact tcgacctgga catgctgccc gccgatgccc
     4441 tggatgattt tgatctggat atgctccctg ctgacgctct cgatgacttc gatctcgata
     4501 tgctgccagg cggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg
     4561 aggagaaccc tggacctgca ccaggaagta tgagcgagct gattaaggag aacatgcaca
     4621 tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg
     4681 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc
     4741 tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca
     4801 accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg
     4861 agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc
     4921 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc
     4981 ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg
     5041 acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga
     5101 tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg
     5161 gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac gaaacgtacg
     5221 tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca
     5281 agcttaatta acaacgttgc acctgtgcct tctagttgcc agccatctgt tgtttgcccc
     5341 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     5401 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     5461 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     5521 tctatgg
//
