LOCUS       sequence-338178-        6581 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template pDNA to prepare DD-CaVT mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6581)
  AUTHORS   Nakanishi H, Yoshii T, Kawasaki S, Hayashi K, Tsutsui K, Oki C, 
            Tsukiji S, Saito H
  TITLE     Light-controllable RNA-protein devices for translational regulation 
            of synthetic mRNAs in mammalian cells.
  JOURNAL   Cell Chem Biol. 2021 Jan 20. pii: S2451-9456(21)00002-7. doi: 
            10.1016/j.chembiol.2021.01.002.
  PUBMED    33508227
REFERENCE   2  (bases 1 to 6581)
  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: "Cell Chem 
            Biol. 2021 Jan 20. pii: S2451-9456(21)00002-7. doi: 
            10.1016/j.chembiol.2021.01.002."
COMMENT     Sequence Label: pcDNA3.1-ecDHFR(DD)-MS2CP-VPg(FCV)
FEATURES             Location/Qualifiers
     source          1..6581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             923..1396
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILER"
     regulatory      1415..1424
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2129..2152
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2172..2189)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2178..2402
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2448..2876
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2535..2554)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2745..2766
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2885..2905)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2890..3219
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3070..3205
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3132..3151
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3286..4080
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3340..3359)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3950..3969
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4254..4375
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4291..4310)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4345..4364
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4424..4440)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4424..4440)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4437..4459)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4448..4464
                     /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(4472..4478,4479..4496,4497..4502))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4517..4538
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4655..4672)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4826..5414)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4906..4925)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5585..6376,6377..6445))
                     /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     6208..6227
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6446..6550)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatgatcag tctgattgcg gcgttagcgg tagattacgt
      961 tatcggcatg gaaaacgcca tgccgtggaa cctgcctgcc gatctcgcct ggtttaaacg
     1021 caacacctta aataaacccg tgattatggg ccgccatacc tgggaatcaa tcggtcgtcc
     1081 gttgccagga cgcaaaaata ttatcctcag cagtcaaccg agtacggacg atcgcgtaac
     1141 gtgggtgaag tcggtggatg aagccatcgc ggcgtgtggt gacgtaccag aaatcatggt
     1201 gattggcggc ggtcgcgtta ttgaacagtt cttgccaaaa gcgcaaaaac tgtatctgac
     1261 gcatatcgac gcagaagtgg aaggcgacac ccatttcccg gattacgagc cggatgactg
     1321 ggaatcggta ttcagcgaat tccacgatgc tgatgcgcag aactctcaca gctattgctt
     1381 tgagattctg gagcggggta ccgagctcgg atctgccacc atggcttcta actttactca
     1441 gttcgttctc gtcgacaatg gcggaactgg cgacgtgact gtcgccccaa gcaacttcgc
     1501 taacggggtc gctgaatgga tcagctctaa ctcgcgatca caggcttaca aagtaacctg
     1561 tagcgttcgt cagagctctg cgcagaatcg caaatacacc atcaaagtcg aggtgcctaa
     1621 aggcgcatgg aggtcttact taaatatgga actaaccatt ccaattttcg ccacgaattc
     1681 cgactgcgag cttattgtta aggcaatgca aggtctccta aaagatggaa acccgattcc
     1741 ctcggccatc gcggccaact ccggcatcta cggatccgcc aagggcaaga ccaagagcaa
     1801 agtgggcccc tacagaggca gaggcgtggc ccttacagac gacgagtatg acgaatggcg
     1861 cgagcacaac gccaccagaa agctggatct gagcgtggaa gatttcctga tgctgcggca
     1921 cagagccgct ctgggagctg atgatgccga cgccgtgaag ttcagatcct ggtggaacag
     1981 cagaagccgg ctggccgacg attacgagga tgtgaccgtg atcggcaaag gcggcgtgaa
     2041 gcacgagaag atccggacca atactctgag agccgtggac agaggctacg acgtgtcctt
     2101 cgctgaagaa accggtgttc caactgttga ttacaaggat gacgacgata agtgataaac
     2161 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2221 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2281 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2341 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2401 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     2461 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2521 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2581 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2641 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2701 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2761 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2821 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     2881 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     2941 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3001 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3061 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3121 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     3181 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     3241 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     3301 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3361 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3421 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     3481 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3541 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3601 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3661 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3721 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     3781 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     3841 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     3901 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     3961 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4021 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4081 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4141 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4201 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     4261 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4321 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4381 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4441 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4501 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4561 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4621 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4681 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4741 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4801 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4861 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4921 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4981 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5041 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5101 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5161 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5221 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5281 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5341 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5401 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5461 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5521 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5581 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5641 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5701 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5761 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5821 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5881 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5941 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6001 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6061 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6121 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6181 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6241 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6301 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6361 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6421 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6481 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6541 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
