LOCUS       sequence-351693-        5434 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of miRFP670-eDHFR(69K6).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5434)
  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 5434)
  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: pCMV-miRFP670-eDHFR(69K6)
FEATURES             Location/Qualifiers
     source          1..5434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        134..437
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        438..641
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     592..612
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      676..685
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             682..1626
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     polyA_signal    2295..2416
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2332..2351)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2386..2405
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2423..2878)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2560..2581)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2772..2791
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2905..3009
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3011..3368
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3011..3202
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3034..3054)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3219..3354
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3403..4197
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3403..4197
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3457..3476)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4067..4086
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4385..4404)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4429..4476
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4805..5393
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5294..5313
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
       61 taccgccatg cattagttat taatagtaat caattacggg gtcattagtt catagcccat
      121 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      181 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      241 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      301 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      361 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      421 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      481 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      541 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      601 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga
      661 tccgctagcc aattggccac catggtagca ggtcatgcct ctggcagccc cgcattcggg
      721 accgcctctc attcgaattg cgaacatgaa gagatccacc tcgccggctc gatccagccg
      781 catggcgcgc ttctggtcgt cagcgaacat gatcatcgcg tcatccaggc cagcgccaac
      841 gccgcggaat ttctgaatct cggaagcgta ctcggcgttc cgctcgccga gatcgacggc
      901 gatctgttga tcaagatcct gccgcatctc gatcccaccg ccgaaggcat gccggtcgcg
      961 gtgcgctgcc ggatcggcaa tccctctacg gagtactgcg gtctgatgca tcggcctccg
     1021 gaaggcgggc tgatcatcga actcgaacgt gccggcccgt cgatcgatct gtcaggcacg
     1081 ctggcgccgg cgctggagcg gatccgcacg gcgggttcac tgcgcgcgct gtgcgatgac
     1141 accgtgctgc tgtttcagca gtgcaccggc tacgaccggg tgatggtgta tcgtttcgat
     1201 gagcaaggcc acggcctggt attctccgag tgccatgtgc ctgggctcga atcctatttc
     1261 ggcaaccgct atccgtcgtc gactgtcccg cagatggcgc ggcagctgta cgtgcggcag
     1321 cgcgtccgcg tgctggtcga cgtcacctat cagccggtgc cgctggagcc gcggctgtcg
     1381 ccgctgaccg ggcgcgatct cgacatgtcg ggctgcttcc tgcgctcgat gtcgccgtgc
     1441 catctgcagt tcctgaagga catgggcgtg cgcgccaccc tggcggtgtc gctggtggtc
     1501 ggcggcaagc tgtggggcct ggttgtctgt caccattatc tgccgcgctt catccgtttc
     1561 gagctgcggg cgatctgcaa acggctcgcc gaaaggatcg cgacgcggat caccgcgctt
     1621 gagagcctct acaagtctgg gtccgcagcc agtagcgccg gaagtatgat cagtctgatt
     1681 gcggcgttag cggtagatcg cgttatcggc atggaaaacg ccatgccgtg gaacctgcct
     1741 gccgatctcg cctggtttaa acgcaacacc ttaaataaac ccgtgattat gggccgccat
     1801 acctgggaat caatcggtcg tccgttgcca ggacgcaaaa atattatcct cagcagtcaa
     1861 ccgggtacgg acaaaaaaaa gaaaaagaaa gatcgcgtaa cgtgggtgaa gtcggtggat
     1921 gaagccatcg cggcgtgtgg tgacgtacca gaaatcatgg tgattggcgg cggtcgcgtt
     1981 tatgaacagt tcttgccaaa agcgcaaaaa ctgtatctga cgcatatcga cgcagaagtg
     2041 gaaggcgaca cccatttccc ggattacgag ccggatgact gggaatcggt attcagcgaa
     2101 ttccacgatg ctgatgcgca gaactctcac agctattgct ttgagattct ggagcggcgg
     2161 ctgtacaagt aaagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg
     2221 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     2281 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     2341 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     2401 tcatcaatgt atcttaaggc gtaaattgta agcgttaata ttttgttaaa attcgcgtta
     2461 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     2521 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     2581 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     2641 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     2701 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     2761 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     2821 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca
     2881 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2941 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3001 aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa
     3061 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3121 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3181 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3241 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3301 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3361 tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     3421 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     3481 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3541 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg
     3601 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     3661 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3721 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3781 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     3841 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     3901 ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg
     3961 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4021 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4081 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4141 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     4201 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     4261 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4321 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc
     4381 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa
     4441 gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg
     4501 gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt
     4561 ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg
     4621 gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac
     4681 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4741 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4801 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4861 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4921 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     4981 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5041 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5101 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5161 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5221 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5281 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5341 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5401 gcaacgcggc ctttttacgg ttcctggcct tttg
//
