LOCUS       sequence-181445-        7121 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of B2AR-APEX in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7121)
  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 7121)
  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-B2AR-APEX
FEATURES             Location/Qualifiers
     source          1..7121
                     /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             2013..2759
                     /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"
     CDS             2760..2795
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     primer_bind     complement(2839..2856)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2845..3069
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3202..3221)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3353..3400
                     /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             3408..4430
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3408..4430
                     /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    4560..4681
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4597..4616)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4651..4670
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4730..4746)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4730..4746)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4743..4765)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4754..4770
                     /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(4778..4784,4785..4802,4803..4808))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4823..4844
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4961..4978)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5132..5720)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5212..5231)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5891..6682,6683..6751))
                     /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     6514..6533
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6752..6856)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6931..6950)
                     /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 tggggcaacc tggcaatgga tcagcttttc
      781 tcctcgcccc taatcggagc cacgctcccg accacgatgt gactcagcag agagacgagg
      841 tctgggttgt aggcatgggt attgtgatgt cactgatcgt cctggcaatt gtgttcggca
      901 acgtcctcgt cattactgct attgcaaagt tcgagcggct tcagacggtt accaattact
      961 ttattacttc cctcgcctgt gccgacttgg tcatgggcct tgcggttgta ccgtttggcg
     1021 cagctcatat cctgatgaag atgtggactt ttggtaattt ttggtgcgaa ttctggactt
     1081 caattgacgt gctctgcgtc acagcgtcaa tcgaaactct gtgtgttatt gccgttgata
     1141 ggtacttcgc gattacgagt ccctttaaat accagtctct gctgactaag aacaaagctc
     1201 gggttatcat actcatggtg tggatcgtct caggactcac aagctttctg cctatacaga
     1261 tgcactggta tcgcgccaca caccaagaag ctatcaattg ttacgcaaac gaaacctgtt
     1321 gcgacttctt cactaatcaa gcctatgcta tcgctagttc aattgtctct ttttatgtgc
     1381 ctctggttat catggtgttc gtctactctc gggtttttca ggaggcaaaa agacagctcc
     1441 agaaaatcga caagtcagaa ggccgatttc acgttcaaaa tctgagccag gtcgaacaag
     1501 acggaagaac tggacatggt cttcggagat cttccaaatt ttgcttgaag gaacacaagg
     1561 ccctgaagac cttggggata attatgggca cattcactct ctgctggctc ccctttttca
     1621 ttgtcaatat cgttcacgtt atacaagaca acttgatcag aaaagaggtg tatatcctcc
     1681 tgaactggat tggctacgtg aactctgggt tcaaccctct gatttactgc cgcagcccag
     1741 acttcagaat cgcattccag gaattgctgt gtctgaggcg ctcttccttg aaggcttatg
     1801 gaaacggata ctcctctaat ggcaacacgg gcgaacagtc aggataccac gtggagcagg
     1861 aaaaagagaa taagctgttg tgcgaggacc tccctggcac tgaggatttt gttggccacc
     1921 aaggaacagt cccaagcgac aatattgaca gccaggggcg aaactgcagc actaatgatt
     1981 cactgctggg tggaagttct ggcggttcaa gtggaaagtc ttacccaact gtgagtgctg
     2041 attaccagga cgccgttgag aaggcgaaga agaagctcag aggcttcatc gctgagaaga
     2101 gatgcgctcc tctaatgctc cgtttggcat tccactctgc tggaaccttt gacaagggca
     2161 cgaagaccgg tggacccttc ggaaccatca agcaccctgc cgaactggct cacagcgcta
     2221 acaacggtct tgacatcgct gttaggcttt tggagccact caaggcggag ttccctattt
     2281 tgagctacgc cgatttctac cagttggctg gcgttgttgc cgttgaggtc acgggtggac
     2341 ctaaggttcc attccaccct ggaagagagg acaagcctga gccaccacca gagggtcgct
     2401 tgcccgatcc cactaagggt tctgaccatt tgagagatgt gtttggcaaa gctatggggc
     2461 ttactgacca agatatcgtt gctctatctg ggggtcacac tattggagct gcacacaagg
     2521 agcgttctgg atttgagggt ccctggacct ctaatcctct tattttcgac aactcatact
     2581 tcacggagtt gttgagtggt gagaaggaag gtctccttca gctaccttct gacaaggctc
     2641 ttttgtctga ccctgtattc cgccctctcg ttgacaaata tgcagcggac gaagatgcct
     2701 tctttgctga ttacgctgag gctcaccaaa agctttccga gcttgggttt gctgatgccg
     2761 aagatcaggt ggatccgcgc ctgattgatg gcaaataata gctcgagtct agagggcccg
     2821 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     2881 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     2941 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     3001 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     3061 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     3121 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3181 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3241 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3301 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc tagaagttcc
     3361 tattccgaag ttcctattct ctagaaagta taggaacttc cttggccaaa aagcctgaac
     3421 tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga
     3481 tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat
     3541 atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc
     3601 actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga
     3661 gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa
     3721 ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg
     3781 atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta
     3841 catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga
     3901 tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg
     3961 aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga
     4021 cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc
     4081 aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga
     4141 cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata
     4201 tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg
     4261 cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc
     4321 gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg
     4381 ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtactac
     4441 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4501 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4561 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4621 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4681 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     4741 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4801 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4861 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4921 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4981 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5041 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5101 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5161 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5221 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5281 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5341 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5401 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5461 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5521 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5581 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5641 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5701 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5761 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5821 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5881 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5941 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6001 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6061 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6121 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6181 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6241 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6301 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6361 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6421 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6481 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6541 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6601 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6661 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6721 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6781 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6841 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     6901 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     6961 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
     7021 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
     7081 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt t
//
