LOCUS       sequence-199684-        7696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AT2R with FLAG tag and CFP in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7696)
  AUTHORS   Inuzuka T, Fujioka Y, Tsuda M, Fujioka M, Satoh AO, Horiuchi K, 
            Nishide S, Nanbo A, Tanaka S, Ohba Y
  TITLE     Attenuation of ligand-induced activation of angiotensin II type 1 
            receptor signaling by the type 2 receptor via protein kinase C.
  JOURNAL   Sci Rep. 2016 Feb 9;6:21613. doi: 10.1038/srep21613.
  PUBMED    26857745
REFERENCE   2  (bases 1 to 7696)
  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: "Sci Rep."; 
            date: "2016-02-9"; volume: "6:21613. doi"; pages: " 
            10.1038/srep21613"
COMMENT     Sequence Label: pCXN2-FLAG-AT2R-CFP
FEATURES             Location/Qualifiers
     source          1..7696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..660
                     /label=chicken beta-actin promoter
     intron          661..1673
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1597..1619
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1681..1700
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1734..1757
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(2853..2855,2856..2858,2859..3371)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIE"
     primer_bind     complement(2898..2919)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3159..3178)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3506..3527
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3568..3587)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3633..3688
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3687..3706)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4049..4065)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4049..4065)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4062..4084)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4073..4089
                     /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(4097..4103,4104..4121,4122..4127))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4142..4163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4222..4417
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4268..4403
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4423..4557
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4460..4479)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4514..4533
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4625..4642)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4796..5384)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4876..4895)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5555..6346,6347..6415))
                     /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     6178..6197
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6416..6520)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6832..7635
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6895..6914)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7505..7524
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    7642..7690
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg gcgagggcgg
      541 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      601 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      661 ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc
      721 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      781 ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct gcgtgaaagc
      841 cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg gtgcgtgcgt
      901 gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg tgagcgctgc
      961 gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg gccgggggcg
     1021 gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt
     1081 gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc ctgcaccccc
     1141 ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg gggcgtggcg
     1201 cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc
     1261 cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct
     1441 ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc
     1501 ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg
     1561 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     1621 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
     1681 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattcacc atggactaca
     1741 aagacgatga cgacaagaag ggcaactcca cccttgccac tactagcaaa aacattacca
     1801 gcggtcttca cttcgggctt gtgaacatct ctggcaacaa tgagtctacc ttgaactgtt
     1861 cacagaaacc atcagataag catttagatg caattcctat tctttactac attatatttg
     1921 taattggatt tctggtcaat attgtcgtgg ttacactgtt ttgttgtcaa aagggtccta
     1981 aaaaggtttc tagcatatac atcttcaacc tcgctgtggc tgatttactc cttttggcta
     2041 ctcttcctct atgggcaacc tattattctt atagatatga ctggctcttt ggacctgtga
     2101 tgtgcaaagt ttttggttct tttcttaccc tgaacatgtt tgcaagcatt ttttttatca
     2161 cctgcatgag tgttgatagg taccaatctg tcatctaccc ctttctgtct caaagaagaa
     2221 atccctggca agcatcttat atagttcccc ttgtttggtg tatggcctgt ttgtcctcat
     2281 tgccaacatt ttattttcga gacgtcagaa ccattgaata cttaggagtg aatgcttgca
     2341 ttatggcttt cccacctgag aaatatgccc aatggtcagc tgggattgcc ttaatgaaaa
     2401 atatccttgg ttttattatc cctttaatat tcatagcaac atgctatttt ggaattagaa
     2461 aacacttact gaagacgaat agctatggga agaacaggat aacccgtgac caagtcctga
     2521 agatggcagc tgctgttgtt ctggccttca tcatttgctg gcttcccttc catgttctga
     2581 ccttcctgga tgctctggcc tggatgggtg tcattaatag ctgcgaagtt atagcagtca
     2641 ttgacctggc acttcctttt gccatcctct tgggattcac caacagctgc gttaatccgt
     2701 ttctgtattg ttttgttgga aaccggttcc aacagaagct ccgcagtgtg tttagggttc
     2761 caattacttg gctccaaggg aaaagagaga gtatgtcttg ccggaaaagc agttctctta
     2821 gagaaatgga gacctttgtg tcttgcggcc gcatggtgag caagggcgag gagctgttca
     2881 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aggttcagcg
     2941 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     3001 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tggggcgtgc
     3061 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     3121 ccgaaggcta cgtccaggag cgtaccatct tcttcaagga cgacggcaac tacaagaccc
     3181 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     3241 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac atcagccaca
     3301 acgtctatat caccgccgac aagcagaaga acggcatcaa ggcccacttc aagatccgcc
     3361 acaacatcga ggacggcggc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     3421 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     3481 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     3541 tcactctcgg catggacgag ctgtagatct ttttccctct gccaaaaatt atggggacat
     3601 catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat
     3661 agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt
     3721 aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc
     3781 catgaacaaa ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca
     3841 ttccttattc catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt
     3901 gttatttttt tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt
     3961 cctcctctcc tgactactcc cagtcatagc tgtccctctt ctcttatgga gatccctcga
     4021 cctgcagccc aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4081 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4141 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4201 acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca accatagtcc cgcccctaac
     4261 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4321 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     4381 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct
     4441 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4501 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatccgc
     4561 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4621 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4681 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4741 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4801 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4861 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4921 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4981 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5041 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5101 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5161 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5221 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5281 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5341 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5401 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5461 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5521 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5581 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5641 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5701 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5761 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5821 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5881 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5941 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6001 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6061 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6121 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6181 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6241 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6301 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6361 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6421 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6481 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6541 cacctgggtc gagcagtgtg gttttcaaga ggaagcaaaa agcctctcca cccaggcctg
     6601 gaatgtttcc acccaatgtc gagcagtgtg gttttgcaag aggaagcaaa aagcctctcc
     6661 acccaggcct ggaatgtttc cacccaatgt cgagcaaacc ccgcccagcg tcttgtcatt
     6721 ggcgaattaa ttcgaacacg cagatgcagt cggggcggcg cggtcccagg tccacttcgc
     6781 atattaaggt gacgcgtgtg gcctcgaaca ccgagcgacc ctgcagccaa tatgggatcg
     6841 gccattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     6901 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     6961 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     7021 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     7081 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     7141 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     7201 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     7261 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     7321 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     7381 ggcgatgatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     7441 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     7501 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     7561 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     7621 gacgagttct tctgagggga tcggcaataa aaagacagaa taaaacgcac gggtgttggg
     7681 tcgtttgttc ggatcc
//
