LOCUS       sequence-181447-        6923 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of AT1R-APEX in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6923)
  AUTHORS   Paek J, Kalocsay M, Staus DP, Wingler L, Pascolutti R, Paulo JA, 
            Gygi SP, Kruse AC
  TITLE     Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed
            Proximity Labeling.
  JOURNAL   Cell. 2017 Apr 6;169(2):338-349.e11. doi: 
            10.1016/j.cell.2017.03.028.
  PUBMED    28388415
REFERENCE   2  (bases 1 to 6923)
  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"; doi: 
            "10.1016/j.cell.2017.03"; journalName: "Cell"; date: "2017-04-6- 6";
            volume: "169"; issue: "2"; pages: "338-349.e11"
COMMENT     Sequence Label: pcDNA5/FRT/TO-AT1R-APEX
FEATURES             Location/Qualifiers
     source          1..6923
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1851..2597
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     primer_bind     complement(2641..2658)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2647..2871
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3004..3023)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3155..3202
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3210..4232
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3210..4232
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4362..4483
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4399..4418)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4453..4472
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4532..4548)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4532..4548)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4545..4567)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4556..4572
                     /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(4580..4586,4587..4604,4605..4610))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4625..4646
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4763..4780)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4934..5522)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5014..5033)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5693..6484,6485..6553))
                     /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     6316..6335
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6554..6658)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6733..6752)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagctta tgattctcaa ctcttctact gaagatggta
      781 ttaaaagaat ccaagatgat tgtcccaaag ctggaaggca taattacata tttgtcatga
      841 ttcctacttt atacagtatc atctttgtgg tgggaatatt tggaaacagc ttggtggtga
      901 tagtcattta cttttatatg aagctgaaga ctgtggccag tgtttttctt ttgaatttag
      961 cactggctga cttatgcttt ttactgactt tgccactatg ggctgtctac acagctatgg
     1021 aataccgctg gccctttggc aattacctat gtaagattgc ttcagccagc gtcagtttca
     1081 acctgtacgc tagtgtgttt ctactcacgt gtctcagcat tgatcgatac ctggctattg
     1141 ttcacccaat gaagtcccgc cttcgacgca caatgcttgt agccaaagtc acctgcatca
     1201 tcatttggct gctggcaggc ttggccagtt tgccagctat aatccatcga aatgtatttt
     1261 tcattgagaa caccaatatt acagtttgtg ctttccatta tgagtcccaa aattcaaccc
     1321 ttccgatagg gctgggcctg accaaaaata tactgggttt cctgtttcct tttctgatca
     1381 ttcttacaag ttatactctt atttggaagg ccctaaagaa ggcttatgaa attcagaaga
     1441 acaaaccaag aaatgatgat atttttaaga taattatggc aattgtgctt ttctttttct
     1501 tttcctggat tccccaccaa atattcactt ttctggatgt attgattcaa ctaggcatca
     1561 tacgtgactg tagaattgca gatattgtgg acacggccat gcctatcacc atttgtatag
     1621 cttattttaa caattgcctg aatcctcttt tttatggctt tctggggaaa aaatttaaaa
     1681 gatattttct ccagcttcta aaatatattc ccccaaaagc caaatcccac tcaaaccttt
     1741 caacaaaaat gagcacgctt tcctaccgcc cctcagataa tgtaagctca tccaccaaga
     1801 agcctgcacc atgttttgag gttgagggtg gaagttctgg cggttcaagt ggaaagtctt
     1861 acccaactgt gagtgctgat taccaggacg ccgttgagaa ggcgaagaag aagctcagag
     1921 gcttcatcgc tgagaagaga tgcgctcctc taatgctccg tttggcattc cactctgctg
     1981 gaacctttga caagggcacg aagaccggtg gacccttcgg aaccatcaag caccctgccg
     2041 aactggctca cagcgctaac aacggtcttg acatcgctgt taggcttttg gagccactca
     2101 aggcggagtt ccctattttg agctacgccg atttctacca gttggctggc gttgttgccg
     2161 ttgaggtcac gggtggacct aaggttccat tccaccctgg aagagaggac aagcctgagc
     2221 caccaccaga gggtcgcttg cccgatccca ctaagggttc tgaccatttg agagatgtgt
     2281 ttggcaaagc tatggggctt actgaccaag atatcgttgc tctatctggg ggtcacacta
     2341 ttggagctgc acacaaggag cgttctggat ttgagggtcc ctggacctct aatcctctta
     2401 ttttcgacaa ctcatacttc acggagttgt tgagtggtga gaaggaaggt ctccttcagc
     2461 taccttctga caaggctctt ttgtctgacc ctgtattccg ccctctcgtt gacaaatatg
     2521 cagcggacga agatgccttc tttgctgatt acgctgaggc tcaccaaaag ctttccgagc
     2581 ttgggtttgc tgatgcctaa tagctcgagt ctagagggcc cgtttaaacc cgctgatcag
     2641 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     2701 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     2761 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2821 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     2881 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     2941 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3001 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3061 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3121 aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt
     3181 ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg
     3241 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg
     3301 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
     3361 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc
     3421 tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct
     3481 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
     3541 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
     3601 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat
     3661 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
     3721 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc
     3781 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
     3841 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
     3901 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
     3961 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg
     4021 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
     4081 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
     4141 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
     4201 gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg
     4261 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4321 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4381 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4441 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4501 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4561 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4621 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4681 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4741 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4801 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4861 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4921 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4981 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5041 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5101 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5161 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5221 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5281 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5341 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5401 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5461 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5521 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5581 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5641 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5701 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5761 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5821 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5881 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5941 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6001 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6061 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6121 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6181 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6241 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6301 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6361 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6421 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6481 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6541 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6601 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6661 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     6721 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     6781 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     6841 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     6901 tgtacgggcc agatatacgc gtt
//
