LOCUS       sequence-492551-        5076 bp    DNA     circular SYN 11-NOV-2025
DEFINITION  Expression plasmid for rtTA and PuroR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5076)
  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 5076)
  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: CMV-rtTA-BG-CMV-PuroR-BGH_pKG1701
FEATURES             Location/Qualifiers
     source          1..5076
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        11..115
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(116..184,185..976)
                     /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(334..353)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1147..1735
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1636..1655
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1817..1851
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1888..1912
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        1959..2262
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2263..2466
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             2492..3238
                     /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"
     primer_bind     complement(3329..3348)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3394..3449
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3448..3467)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    complement(3587..3611)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        3644..3947
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3948..4151
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             4178..4777
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4178..4197)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4674..4694
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4778..5002
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5011..5073
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 ctataatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
       61 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
      121 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
      181 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
      241 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
      301 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
      361 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
      421 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
      481 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
      541 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
      601 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
      661 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
      721 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
      781 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
      841 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
      901 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
      961 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     1021 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     1081 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     1141 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     1201 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     1261 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     1321 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     1381 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     1441 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     1501 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     1561 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     1621 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     1681 ctgacttgag cgtcgatttt tgtgatgctc gtcaggggtg cggagcctat ggaaaaacgc
     1741 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     1801 tcctgcgtta tcttcaccct agaaagatag tctgcgtaaa attgacgcat gtccttactc
     1861 atagcaggtg ccactagtgg agttccgaca agtttgtaca aaaaagcagg ctccaccatg
     1921 ggaaccaatt cagtcgactg gatccggtac aggatccgcg ttacataact tacggtaaat
     1981 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     2041 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     2101 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     2161 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     2221 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     2281 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     2341 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     2401 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     2461 agagctcgtt aactctaaaa agaatgtcga catgagccgg ctggacaaga gcaaagtgat
     2521 caacagcgcc ctggaactgc tgaacggcgt gggcatcgag ggcctgacca cccggaagct
     2581 ggcccagaaa ctgggcgtgg aacagcccac cctgtactgg cacgtgaaga acaagcgggc
     2641 cctgctggac gccctgccca tcgagatgct ggaccggcac cacacccaca gctgccctct
     2701 ggaaggcgag agctggcagg acttcctgcg gaacaacgcc aagagctaca gatgcgccct
     2761 gctgagccac cgggacggcg ccaaagtgca cctgggcacc agacccaccg agaagcagta
     2821 cgagacactg gaaaaccagc tggccttcct gtgccagcag ggcttcagcc tggaaaacgc
     2881 cctgtacgcc ctgagcgccg tgggccactt caccctgggc tgcgtgctgg aagaacagga
     2941 acaccaggtc gccaaagagg aaagagagac acccaccacc gacagcatgc cccccctgct
     3001 gaagcaggcc atcgagctgt tcgacagaca gggcgccgag cccgccttcc tgttcggcct
     3061 ggaactgatc atctgcggcc tcgagaagca gctgaagtgc gagagcggcg gacccaccga
     3121 cgccctggac gacttcgacc tggacatgct gcccgccgat gccctggatg attttgatct
     3181 ggatatgctc cctgctgacg ctctcgatga cttcgatctc gatatgctgc caggctaaaa
     3241 cgcgtgaatt cactcctcag gtgcaggctg cctatcagaa ggtggtgact ggtgtggcca
     3301 atgccctggc tcacaaatac cactgagatc tttttccctc tgccaaaaat tatggggaca
     3361 tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa
     3421 tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt
     3481 taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat atgctggctg
     3541 ccatgaacaa aggttggcta taaagaggtc atcatcggat atctagaccc agctttcttg
     3601 tacaaagtgg tcacctgcta tgcgctgata taggtaggct actcgttaca taacttacgg
     3661 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     3721 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     3781 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     3841 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     3901 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     3961 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     4021 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     4081 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     4141 taagcagagc tgagctcgtt tagtgaaccg tcagatgatg accgagtaca agcccacggt
     4201 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
     4261 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
     4321 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     4381 cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     4441 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     4501 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     4561 cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     4621 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     4681 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     4741 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgactg tgccttctag ttgccagcca
     4801 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4861 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4921 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     4981 ggggatgcgg tgggctctat ggagcttaca catgcgtcaa ttttacgcat gattatcttt
     5041 aacgtacgtc acaatatgat tatctttcta gggtta
//
