LOCUS       sequence-388602-        7373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV driven expression of the consitutively active calcium recorder 
            positive control Caprola_on in mammalian cell lines fused to mEGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7373)
  AUTHORS   Huppertz MC, Wilhelm J, Grenier V, Schneider MW, Falt T, Porzberg N,
            Hausmann D, Hoffmann DC, Hai L, Tarnawski M, Pino G, Slanchev K, 
            Kolb I, Acuna C, Fenk LM, Baier H, Hiblot J, Johnsson K
  TITLE     Recording physiological history of cells with chemical labeling.
  JOURNAL   Science. 2024 Feb 23;383(6685):890-897. doi: 
            10.1126/science.adg0812. Epub 2024 Feb 22.
  PUBMED    38386755
REFERENCE   2  (bases 1 to 7373)
  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: 
            "10.1126/science.adg0812"; journalName: "Science"; date: 
            "2024-02-23- 23"; volume: "383"; issue: "6685"; pages: "890-897"
COMMENT     Sequence Label: pCDNA5/FRT_CMV_NES-Caprola_on-mEGFP
FEATURES             Location/Qualifiers
     source          1..7373
                     /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             1016..1456
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAEL
                     RHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVVMMTAK"
     CDS             join(2573..2575,2576..2578,2579..3292)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2618..2639)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2879..2898)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3226..3247
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3324..3341)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3330..3554
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3687..3706)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3838..3885
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3893..4915
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3893..4915
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5045..5166
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5082..5101)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5136..5155
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5215..5231)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5215..5231)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5228..5250)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5239..5255
                     /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(5263..5269,5270..5287,5288..5293))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5308..5329
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5446..5463)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5617..6206)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5698..5717)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6377..7168,7169..7237))
                     /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     7000..7019
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7238..7342)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctcactg 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 accaccatgc tgcagaacga gcttgctctt aagttggctg gacttgatat
      961 taacaagact ggaggttctg cccgcgagac cttccaggcc ttccgcaccg gttctgacca
     1021 actgactgaa gagcagatcg cagaatttaa agaggctttc tccctatttg acaaggacgg
     1081 ggatgggaca ataacaacca aggagctggg gacggtgatg cggtctctgg ggcagaaccc
     1141 cacagaagca gagctgcagg acatgatcaa tgaagtagat gccgacggtg acggcacaat
     1201 cgacttccct gagttcctga caatgatggc aagaaaaatg aaagacacag acagtgaaga
     1261 agaaattaga gaagcgttcc gtgtgtttga taaggatggc aatggctaca tcagtgcagc
     1321 agagcttcgc cacgtgatga caaaccttgg agagaagtta acagatgaag aggttgatga
     1381 aatgatcagg gaagcagaca tcgatgggga tggtcaggta aactacgaag agtttgtagt
     1441 gatgatgaca gcgaaggaat tccctcctcc tccgccaccg ccaccaccgc cgccgccgcc
     1501 accaccacct cctccgcctc ctccaccacc accgccaccg cctccaccac ctggcggcag
     1561 cagggtcgac tcatcacgtc gtaagttcaa taagacaggt cacgcactga gagctatagg
     1621 tcggctgagc tcactcgagg gcggttcttt tgcccgcgag accttccagg ccttccgcac
     1681 caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc
     1741 gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg agccgttcct
     1801 gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa tcgccggtga
     1861 gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc agtcccctgt
     1921 cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg aagccgctcg
     1981 cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc tgaatctgct
     2041 gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt ctactctgga
     2101 gattggtggc accggtggct ctggcggtac cggtggctct ggcggttcta tcggtactgg
     2161 ctttccattc gacccccatt atgtggaagt cctgggcgag cgcatgcact acgtcgatgt
     2221 tggtccgcgc gatggcaccc ctgtgctgtt cctgcacggt aacccgacct cctcctacgt
     2281 gtggcgcaac atcatcccgc atgttgcacc gacccatcgc tgcattgctc cagacctgat
     2341 cggtatgggc aaatccgaca aaccagacct gggttatttc ttcgacgacc acgtccgctt
     2401 catggatgcc ttcatcgaag ccctgggtct ggaagaggtc gtcctggtca ttcacgactg
     2461 gggctccgct ctgggtttcc actgggccaa gcgcaatcca gagcgcgtca aaggtattgc
     2521 atttatggag ttcatccgcc ctatcccgac ctgggacgaa tggggaggtt ctatggtgag
     2581 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     2641 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2701 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2761 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2821 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2881 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2941 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     3001 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     3061 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     3121 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     3181 cacccagtcc aaactgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3241 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aatctagagg
     3301 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3361 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3421 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     3481 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3541 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3601 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3661 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3721 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3781 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa
     3841 gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc
     3901 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     3961 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     4021 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     4081 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     4141 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     4201 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     4261 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     4321 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     4381 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     4441 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     4501 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     4561 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     4621 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     4681 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     4741 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     4801 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     4861 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt
     4921 actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4981 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     5041 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5101 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5161 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     5221 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5281 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5341 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5401 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5461 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5521 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5581 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gccccccctg
     5641 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5701 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5761 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5821 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5881 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5941 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6001 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6061 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6121 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6181 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6241 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6301 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6361 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6421 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6481 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6541 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6601 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6661 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6721 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6781 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6841 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6901 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6961 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7021 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7081 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7141 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7201 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7261 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7321 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
