LOCUS       sequence-351722-        6351 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of eDHFR(69K6)-iRFP713.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6351)
  AUTHORS   Suzuki S, Nakamura A, Hatano Y, Yoshikawa M, Yoshii T, Sawada S, 
            Atsuta-Tsunoda K, Aoki K, Tsukiji S
  TITLE     A chemogenetic platform for controlling plasma membrane signaling 
            and synthetic signal oscillation.
  JOURNAL   Cell Chem Biol. 2022 Sep 15;29(9):1446-1464.e10. doi: 
            10.1016/j.chembiol.2022.06.005. Epub 2022 Jul 13.
  PUBMED    35835118
REFERENCE   2  (bases 1 to 6351)
  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.chembiol.2022.06.005"; journalName: "Cell Chem Biol"; 
            date: "2022-09-15- 15"; volume: "29"; issue: "9"; pages: 
            "1446-1464.e10"
COMMENT     Sequence Label: pCAGGS-eDHFR(69K6)-iRFP713
FEATURES             Location/Qualifiers
     source          1..6351
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          127..1143
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1067..1089
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1151..1170
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(2719..2739)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2719..2737)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2758..2774)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2876..2895)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2941..2996
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2995..3014)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3357..3373)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3381..3397
                     /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(3405..3411,3412..3429,3430..3435))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3450..3471
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3530..3725
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3576..3711
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3731..3865
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3768..3787)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3822..3841
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3933..3950)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4104..4692)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4184..4203)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4863..5654,5655..5723))
                     /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     5486..5505
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5724..5828)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5859..6238
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
       61 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
      121 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
      181 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
      241 tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg
      301 cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt
      361 gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt
      421 gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg
      481 gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg
      541 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc
      601 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg
      661 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc
      721 ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc
      781 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag
      841 agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc
      901 cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
      961 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc
     1021 tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg
     1081 cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta
     1141 cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattcata
     1201 gtagctagca ccaccatgat cagtctgatt gcggcgttag cggtagatcg cgttatcggc
     1261 atggaaaacg ccatgccgtg gaacctgcct gccgatctcg cctggtttaa acgcaacacc
     1321 ttaaataaac ccgtgattat gggccgccat acctgggaat caatcggtcg tccgttgcca
     1381 ggacgcaaaa atattatcct cagcagtcaa ccgggtacgg acaaaaaaaa gaaaaagaaa
     1441 gatcgcgtaa cgtgggtgaa gtcggtggat gaagccatcg cggcgtgtgg tgacgtacca
     1501 gaaatcatgg tgattggcgg cggtcgcgtt tatgaacagt tcttgccaaa agcgcaaaaa
     1561 ctgtatctga cgcatatcga cgcagaagtg gaaggcgaca cccatttccc ggattacgag
     1621 ccggatgact gggaatcggt attcagcgaa ttccacgatg ctgatgcgca gaactctcac
     1681 agctattgct ttgagattct ggagcggcgg aacgtcggcg gccgctctgg tgctggtagt
     1741 ggcgctggtt ccggtatggc cgaagggagc gtggcaagac agcccgatct gctgacatgc
     1801 gacgacgaac caatccacat tccaggagct atccagcctc atggactgct gctggctctg
     1861 gccgctgaca tgactatcgt ggcaggctct gataacctgc cagagctgac cgggctggct
     1921 atcggagcac tgattggccg gtccgcagcc gacgtgttcg attctgagac tcacaataga
     1981 ctgaccatcg ccctggctga acctggggct gcagtgggag ccccaattac cgtcggcttc
     2041 acaatgcgaa aggacgctgg ctttatcggg tcctggcacc gccatgatca gctgattttt
     2101 ctggagctgg aaccacctca gagagacgtg gcagagccac aggctttctt tcggagaaca
     2161 aacagcgcca tcaggcgcct gcaggccgct gagactctgg aatccgcttg cgcagccgct
     2221 gcacaggaag tgaggaagat caccgggttc gatcgcgtca tgatctacag attcgcctca
     2281 gactttagcg gagaagtgat cgcagaagat cgctgtgccg aggtcgaaag taaactgggc
     2341 ctgcattacc cagcctcaac cgtgcctgca caggcacgac ggctgtatac aatcaacccc
     2401 gtgcggatca ttcccgacat taattacaga ccagtgcccg tcacacctga cctgaaccca
     2461 gtgactggga ggcccatcga tctgagcttc gccattctgc gcagcgtgtc ccctgtccac
     2521 ctggagttta tgaggaatat cggaatgcat ggcaccatgt ctatcagtat tctgagaggc
     2581 gaaaggctgt gggggctgat cgtgtgccac catcggacac cttactatgt ggacctggac
     2641 ggcaggcagg cttgtgaact ggtggctcag gtgctggctt ggcagattgg cgtgatgtag
     2701 gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     2761 atagctgttt cctgtgtgaa attgttatcc gctaattcac tcctcaggtg caggctgcct
     2821 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt
     2881 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct
     2941 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg
     3001 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt
     3061 tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc
     3121 agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag
     3181 gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt
     3241 ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg
     3301 tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca
     3361 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3421 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3481 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt
     3541 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3601 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3661 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3721 gcaaaaagct aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3781 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3841 caatgtatct tatcatgtct ggatccgctg cattaatgaa tcggccaacg cgcggggaga
     3901 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3961 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4021 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4081 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4141 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4201 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4261 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4321 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4381 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4441 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4501 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4561 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4621 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4681 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4741 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4801 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4861 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4921 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4981 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5041 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5101 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5161 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5221 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5281 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5341 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5401 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5461 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5521 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5581 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5641 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5701 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5761 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5821 gttccgcgca catttccccg aaaagtgcca cctgggtcga cattgattat tgactagtta
     5881 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     5941 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     6001 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     6061 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     6121 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     6181 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     6241 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     6301 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg g
//
