LOCUS       sequence-442065-        7644 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid encoding the human Angiotensin II type 
            I receptor fused to the tTA transcriptional activator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7644)
  AUTHORS   Skiba MA, Sterling SM, Rawson S, Zhang S, Xu H, Jiang H, Nemeth GR, 
            Gilman MSA, Hurley JD, Shen P, Staus DP, Kim J, McMahon C, Lehtinen 
            MK, Rockman HA, Barth P, Wingler LM, Kruse AC
  TITLE     Antibodies expand the scope of angiotensin receptor pharmacology.
  JOURNAL   Nat Chem Biol. 2024 May 14. doi: 10.1038/s41589-024-01620-6.
  PUBMED    38744986
REFERENCE   2  (bases 1 to 7644)
  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"; journalName: "Nat Chem 
            Biol. 2024 May 14. doi: 10.1038/s41589-024-01620-6."
COMMENT     Sequence Label: pTarget_AT1R-tTA
FEATURES             Location/Qualifiers
     source          1..7644
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     680..700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1005..1024
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1191..1213
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1229..1248
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1229..1247
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(2462..3076,3077..3463)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="RLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKN
                     KRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPT
                     EKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDS
                     MPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEG
                     LLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADA
                     LDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(2529..2548)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3554..3576)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3565..3581
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3589..3595,3596..3613,3614..3619))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3634..3655
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    3711..3832
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3722..3741)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3776..3795
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3966..4421
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4053..4072)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4263..4284
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4436..4793
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4436..4627
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4459..4479)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4644..4779
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4844..5443
                     /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(4844..4863)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5340..5360
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5507..5555
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5582..5601)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5701..5723
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5761..5779)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5847..5951
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5952..6020,6021..6812)
                     /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(6170..6189)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6983..7571
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7472..7491
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
      661 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaaaattg gatctccatt cgccattcag
     1081 gctgcgcaac tgctgggaag gacgatcaga gcgggcctct tcgctattac gccagctggc
     1141 gaaagggacg tggcaagcaa ggcgattaag ttgagttacg ccaggatttt cccagtcacg
     1201 acgttgtaaa acgacggcca gagaattata atacgactca ctatagggcg aattcggatc
     1261 cttgctagcc gccaccatga agacgatcat cgccctgagc tacatcttct gcctggtatt
     1321 cgccgactac aaggacgatg atgacgccgc cattctcaac tcttctactg aagatggtat
     1381 taaaagaatc caagatgatt gtcccaaagc tggaaggcat aattacatat ttgtcatgat
     1441 tcctacttta tacagtatca tctttgtggt gggaatattt ggaaacagct tggtggtgat
     1501 agtcatttac ttttatatga agctgaagac tgtggccagt gtttttcttt tgaatttagc
     1561 actggctgac ttatgctttt tactgacttt gccactatgg gctgtctaca cagctatgga
     1621 ataccgctgg ccctttggca attacctatg taagattgct tcagccagcg tcagtttcaa
     1681 cctgtacgct agtgtgtttc tactcacgtg tctcagcatt gatcgatacc tggctattgt
     1741 tcacccaatg aagtcccgcc ttcgacgcac aatgcttgta gccaaagtca cctgcatcat
     1801 catttggctg ctggcaggct tggccagttt gccagctata atccatcgaa atgtattttt
     1861 cattgagaac accaatatta cagtttgtgc tttccattat gagtcccaaa attcaaccct
     1921 tccgataggg ctgggcctga ccaaaaatat actgggtttc ctgtttcctt ttctgatcat
     1981 tcttacaagt tatactctta tttggaaggc cctaaagaag gcttatgaaa ttcagaagaa
     2041 caaaccaaga aatgatgata tttttaagat aattatggca attgtgcttt tctttttctt
     2101 ttcctggatt ccccaccaaa tattcacttt tctggatgta ttgattcaac taggcatcat
     2161 acgtgactgt agaattgcag atattgtgga cacggccatg cctatcacca tttgtatagc
     2221 ttattttaac aattgcctga atcctctttt ttatggcttt ctggggaaaa aatttaaaag
     2281 atattttctc cagcttctaa aatatattcc cccaaaagcc aaatcccact caaacctttc
     2341 aacaaaaatg agcacgcttt cctaccgccc ctcagataat gtaagctcat ccaccaagaa
     2401 gcctgcacca tgttttgagg ttgagacttc atcgaccggt gagaacctgt acttccagct
     2461 aagattagat aaaagtaaag tgattaacag cgcattagag ctgcttaatg aggtcggaat
     2521 cgaaggttta acaacccgta aactcgccca gaagctaggt gtagagcagc ctacattgta
     2581 ttggcatgta aaaaataagc gggctttgct cgacgcctta gccattgaga tgttagatag
     2641 gcaccatact cacttttgcc ctttagaagg ggaaagctgg caagattttt tacgtaataa
     2701 cgctaaaagt tttagatgtg ctttactaag tcatcgcgat ggagcaaaag tacatttagg
     2761 tacacggcct acagaaaaac agtatgaaac tctcgaaaat caattagcct ttttatgcca
     2821 acaaggtttt tcactagaga atgcattata tgcactcagc gctgtggggc attttacttt
     2881 aggttgcgta ttggaagatc aagagcatca agtcgctaaa gaagaaaggg aaacacctac
     2941 tactgatagt atgccgccat tattacgaca agctatcgaa ttatttgatc accaaggtgc
     3001 agagccagcc ttcttattcg gccttgaatt gatcatatgc ggattagaaa aacaacttaa
     3061 atgtgaaagt gggtccgcgt acagccgcgc gcgtacgaaa aacaattacg ggtctaccat
     3121 cgagggcctg ctcgatctcc cggacgacga cgcccccgaa gaggcggggc tggcggctcc
     3181 gcgcctgtcc tttctccccg cgggacacac gcgcagactg tcgacggccc ccccgaccga
     3241 tgtcagcctg ggggacgagc tccacttaga cggcgaggac gtggcgatgg cgcatgccga
     3301 cgcgctagac gatttcgatc tggacatgtt gggggacggg gattccccgg gtccgggatt
     3361 taccccccac gactccgccc cctacggcgc tctggatatg gccgacttcg agtttgagca
     3421 gatgtttacc gatgcccttg gaattgacga gtacggtggg taatggtacc gtcgactgcg
     3481 gccgcgaatt ccaagcttga gtattctatc gtgtcaccta aataacttgg cgtaatcatg
     3541 gtcatatctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3601 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     3661 gttgcgcgat gcttccattt tgtgagggtt aatgcttcga gaagacatga taagatacat
     3721 tgatgagttt ggacaaacca caacaagaat gcagtgaaaa aaatgcttta tttgtgaaat
     3781 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     3841 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcaa
     3901 gtaaaacctc tacaaatgtg gtaaaatccg ataaggatcg attccggagc ctgaatggcg
     3961 aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4021 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     4081 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4141 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     4201 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4261 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     4321 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     4381 atttaacgca aattttaaca aaatattaac gcttacaatt tcgcctgtgt accttctgag
     4441 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     4501 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     4561 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4621 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     4681 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     4741 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagcttg
     4801 attcttctga cacaacagtc tcgaacttaa ggctagagcc accatgaccg agtacaagcc
     4861 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     4921 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     4981 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     5041 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     5101 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     5161 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     5221 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     5281 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     5341 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     5401 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgagcgggac tctggggttc
     5461 gaaatgaccg accaagcgac gcccaacctg ccatcacgat ggccgcaata aaatatcttt
     5521 attttcatta catctgtgtg ttggtttttt gtgtgaagat ccgcgtatgg tgcactctca
     5581 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     5641 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     5701 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     5761 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     5821 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5881 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5941 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     6001 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     6061 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     6121 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     6181 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     6241 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     6301 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     6361 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6421 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6481 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6541 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6601 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6661 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6721 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6781 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6841 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6901 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6961 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     7021 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     7081 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     7141 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     7201 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     7261 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     7321 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     7381 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     7441 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7501 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7561 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7621 ttgctcacat ggctcgacag atct
//
