LOCUS       sequence-178097-        6097 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Cal-light M13-TEV-C component and TdTomato in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6097)
  AUTHORS   Lee D, Hyun JH, Jung K, Hannan P, Kwon HB
  TITLE     A calcium- and light-gated switch to induce gene expression in 
            activated neurons.
  JOURNAL   Nat Biotechnol. 2017 Jun 26. doi: 10.1038/nbt.3902.
  PUBMED    28650460
REFERENCE   2  (bases 1 to 6097)
  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: "Nat 
            Biotechnol. 2017 Jun 26. doi: 10.1038/nbt.3902."
COMMENT     Sequence Label: pCMV-M13-TEV-C-P2A-TdTomato
FEATURES             Location/Qualifiers
     source          1..6097
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..978
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1013..1031
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1013..1030
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1066..1083
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1087..1119
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(1123..1131,1132..1146)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     regulatory      1153..1162
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1174..1230
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     CDS             1594..1650
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1657..3087
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     primer_bind     complement(3104..3123)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3105..3123)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3128..3262
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3152..3171)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3206..3225
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3375..3395)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3375..3393)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3414..3430)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3414..3430)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3427..3449)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3438..3454
                     /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(3462..3468,3469..3486,3487..3492))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3507..3528
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3645..3662)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3815..4403)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3895..3914)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4574..5365,5366..5434))
                     /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     5197..5216
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5435..5539)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5565..6020)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5702..5723)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5914..5933
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
       61 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
      121 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
      181 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
      241 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
      301 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
      361 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
      421 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
      481 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
      541 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
      601 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
      661 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
      721 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
      781 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
      841 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
      901 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
      961 aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct tgatttaggt
     1021 gacactatag aatacaagct acttgttctt tttgcaggat ccatgcatca tcatcatcat
     1081 catggtatgg ctagcatgac tggtggacag caaatgggtc gggatctgta cgacgatgac
     1141 gataaggatc tcgccaccat ggtcgactca tcacgtcgta agtggaataa gacaggtcac
     1201 gcagtcagag ctataggtcg gctgagctca ctcgaaggtg gcggtggctc tggaggtggt
     1261 gggtccggag gaggcggcat cgagatgtcc agcatggtga gcgatactag ctgtaccttc
     1321 ccatcatctg acggaatctt ctggaagcac tggattcaga ctaaggacgg ccagtgtggc
     1381 agcccactgg tgagcacacg agacggattc atcgtgggga ttcacagcgc ctccaacttt
     1441 acaaacacca ataactattt cacctcagtg ccaaagaact ttatggagct gctgaccaac
     1501 caggaggccc agcagtgggt gagcgggtgg cgcctgaacg ccgattccgt gctgtggggc
     1561 gggcacaagg tgtttatggc atgcggaagc ggagctacta acttcagcct gctgaagcag
     1621 gctggagacg tggaggagaa ccctggacct gaattcatgg tgagcaaggg cgaggaggtc
     1681 atcaaagagt tcatgcgctt caaggtgcgc atggagggct ccatgaacgg ccacgagttc
     1741 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     1801 gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccccca gttcatgtac
     1861 ggctccaagg cgtacgtgaa gcaccccgcc gacatccccg attacaagaa gctgtccttc
     1921 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggtct ggtgaccgtg
     1981 acccaggact cctccctgca ggacggcacg ctgatctaca aggtgaagat gcgcggcacc
     2041 aacttccccc ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctccacc
     2101 gagcgcctgt acccccgcga cggcgtgctg aagggcgaga tccaccaggc cctgaagctg
     2161 aaggacggcg gccgctacct ggtggagttc aagaccatct acatggccaa gaagcccgtg
     2221 caactgcccg gctactacta cgtggacacc aagctggaca tcacctccca caacgaggac
     2281 tacaccatcg tggaacagta cgagcgctcc gagggccgcc accacctgtt cctggggcat
     2341 ggcaccggca gcaccggcag cggcagttcc ggcaccgcct cctccgagga caacaacatg
     2401 gccgtcatca aagagttcat gcgcttcaag gtgcgcatgg agggctccat gaacggccac
     2461 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     2521 ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc
     2581 atgtacggct ccaaggcgta cgtgaagcac cccgccgaca tccccgatta caagaagctg
     2641 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggtctggtg
     2701 accgtgaccc aggactcctc cctgcaggac ggcacgctga tctacaaggt gaagatgcgc
     2761 ggcaccaact tcccccccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
     2821 tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca ccaggccctg
     2881 aagctgaagg acggcggccg ctacctggtg gagttcaaga ccatctacat ggccaagaag
     2941 cccgtgcaac tgcccggcta ctactacgtg gacaccaagc tggacatcac ctcccacaac
     3001 gaggactaca ccatcgtgga acagtacgag cgctccgagg gccgccacca cctgttcctg
     3061 tacggcatgg acgagctgta caagtagtct agatagctag cctccctata gtgagtcgta
     3121 ttacgtagat ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     3181 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     3241 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     3301 gggggaggtg tgggaggttt tttaattcgc gtccgcggcg ccaatgcatt gggcccggta
     3361 cccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     3421 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccgggagc
     3481 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     3541 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     3601 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     3661 ctgcgctcgg tcgttcggct gcgggagcgg tatcagctca ctcaaaggcg gtaatacggt
     3721 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3781 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3841 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3901 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3961 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4021 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4081 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4141 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4201 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4261 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4321 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4381 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4441 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4501 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4561 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4621 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4681 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4741 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4801 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4861 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4921 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4981 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5041 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5101 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5161 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5221 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5281 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5341 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5401 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5461 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5521 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa
     5581 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     5641 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     5701 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     5761 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     5821 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     5881 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     5941 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     6001 tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg
     6061 atcggtgcgg gcctcttcgc tattacgcca gtcgacc
//
