LOCUS       sequence-388606-        7337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV driven expression of the calcium recorder Caprola_04 in 
            mammalian cell lines fused to mEGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7337)
  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 7337)
  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_04-mEGFP
FEATURES             Location/Qualifiers
     source          1..7337
                     /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             1577..1633
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKFNKTGHAVRAIGRLSS"
     CDS             join(2537..2539,2540..2542,2543..3256)
                     /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(2582..2603)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2843..2862)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3190..3211
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3288..3305)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3294..3518
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3651..3670)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3802..3849
                     /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             3857..4879
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3857..4879
                     /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    5009..5130
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5046..5065)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5100..5119
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5179..5195)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5179..5195)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5192..5214)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5203..5219
                     /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(5227..5233,5234..5251,5252..5257))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5272..5293
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5410..5427)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5581..6170)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5662..5681)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6341..7132,7133..7201))
                     /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     6964..6983
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7202..7306)
                     /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 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 cacgcagtca gagctatagg
     1621 tcggctgagc tcactcgagg gcggttctga cgtcggccgc aagctgatca tcgatcagaa
     1681 cgtttttatc gagggtacgc tgccgatggg tgtcgtccgc ccgctgactg aagtcgagat
     1741 ggaccattac cgcgagccgt tcctgaatcc tgttgaccgc gagccactgt ggcgcttccc
     1801 aaacgagctg ccaatcgccg gtgagccagc gaacatcgtc gcgctggtcg aagaatacat
     1861 ggactggctg caccagtccc ctgtcccgaa gctgctgttc tggggcaccc caggcgttct
     1921 gatcccaccg gccgaagccg ctcgcctggc caaaagcctg cctaactgca aggctgtgga
     1981 catcggcccg ggtctgaatc tgctgcaaga agacaacccg gacctgatcg gcagcgagat
     2041 cgcgcgctgg ctgtctactc tggagattgg tggcaccggt ggctctggcg gtaccggtgg
     2101 ctctggcggt tctatcggta ctggctttcc attcgacccc cattatgtgg aagtcctggg
     2161 cgagcgcatg cactacgtcg atgttggtcc gcgcgatggc acccctgtgc tgttcctgca
     2221 cggtaacccg acctcctcct acgtgtggcg caacatcatc ccacatgttg caccgaccca
     2281 tcgctgcatt gctccagacc tgatcggtat gggcaaatcc gacaaaccag acctgggtta
     2341 tttcttcgac gaccacgtcc gcttcatgga tgccttcatc gaagccctgg gtctggaaga
     2401 ggtcgtcctg gtcattcacg actggggctc cgctctgggt ttccactggg ccaagcgcaa
     2461 tccagagcgc gtcaaaggta ttgcatttat ggagttcatc cgccctatcc cgacctggga
     2521 cgaatgggga ggttctatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2581 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2641 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2701 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     2761 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2821 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2881 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2941 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     3001 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     3061 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     3121 gctgcccgac aaccactacc tgagcaccca gtccaaactg agcaaagacc ccaacgagaa
     3181 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3241 cgagctgtac aagtaatcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     3301 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3361 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3421 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3481 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3541 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3601 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3661 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3721 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3781 gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat
     3841 aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
     3901 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     3961 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     4021 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     4081 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     4141 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
     4201 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     4261 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     4321 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     4381 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     4441 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     4501 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     4561 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     4621 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     4681 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     4741 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     4801 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     4861 tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga
     4921 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4981 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     5041 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     5101 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     5161 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5221 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5281 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5341 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     5401 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5461 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5521 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5581 tttccatagg ctccgccccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5641 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5701 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5761 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5821 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5881 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5941 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6001 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6061 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6121 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6181 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6241 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6301 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6361 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6421 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6481 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6541 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6601 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6661 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6721 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6781 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6841 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6901 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6961 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7021 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7081 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7141 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7201 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7261 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7321 aagtgccacc tgacgtc
//
