LOCUS       sequence-388475-        7306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV driven expression of the inactive calcium recorder negative 
            control Caprola_off in mammalian cell lines fused to mEGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7306)
  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 7306)
  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_off-mEGFP
FEATURES             Location/Qualifiers
     source          1..7306
                     /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             986..1426
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAEL
                     RHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVVMMTAK"
     CDS             1547..1603
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHALRAIGRLSS"
     CDS             join(2507..2509,2510..2512,2513..3226)
                     /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(2552..2573)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2813..2832)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3160..3181
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3258..3275)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3264..3488
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3621..3640)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3772..3819
                     /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             3827..4849
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3827..4849
                     /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    4979..5100
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5016..5035)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5070..5089
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5149..5165)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5149..5165)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5162..5184)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5173..5189
                     /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(5197..5203,5204..5221,5222..5227))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5242..5263
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5380..5397)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5551..6139)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5631..5650)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6310..7101,7102..7170))
                     /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     6933..6952
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7171..7275)
                     /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 ggaggttctc tgccggacca actgactgaa gagcagatcg cagaatttaa
     1021 agaggctttc tccctatttg acaaggacgg ggatgggaca ataacaacca aggagctggg
     1081 gacggtgatg cggtctctgg ggcagaaccc cacagaagca gagctgcagg acatgatcaa
     1141 tgaagtagat gccgacggtg acggcacaat cgacttccct gagttcctga caatgatggc
     1201 aagaaaaatg aaagacacag acagtgaaga agaaattaga gaagcgttcc gtgtgtttga
     1261 taaggatggc aatggctaca tcagtgcagc agagcttcgc cacgtgatga caaaccttgg
     1321 agagaagtta acagatgaag aggttgatga aatgatcagg gaagcagaca tcgatgggga
     1381 tggtcaggta aactacgaag agtttgtagt gatgatgaca gcgaaggaat tccctcctcc
     1441 tccgccaccg ccaccaccgc cgccgccgcc accaccacct cctccgcctc ctccaccacc
     1501 accgccaccg cctccaccac ctggcggcag cagggtcgac tcatcacgtc gtaagtggaa
     1561 taagacaggt cacgcactga gagctatagg tcggctgagc tcactcgagg gcggttctga
     1621 cgtcggccgc aagctgatca tcgatcagaa cgtttttatc gagggtacgc tgccgatggg
     1681 tgtcgtccgc ccgctgactg aagtcgagat ggaccattac cgcgagccgt tcctgaatcc
     1741 tgttgaccgc gagccactgt ggcgcttccc aaacgagctg ccaatcgccg gtgagccagc
     1801 gaacatcgtc gcgctggtcg aagaatacat ggactggctg caccagtccc ctgtcccgaa
     1861 gctgctgttc tggggcaccc caggcgttct gatcccaccg gccgaagccg ctcgcctggc
     1921 caaaagcctg cctaactgca aggctgtgga catcggcccg ggtctgaatc tgctgcaaga
     1981 agacaacccg gacctgatcg gcagcgagat cgcgcgctgg ctgtctactc tggagattgg
     2041 tggcaccggt ggctctggcg gtaccggtgg ctctggcggt tctatcggta ctggctttcc
     2101 attcgacccc cattatgtgg aagtcctggg cgagcgcatg cactacgtcg atgttggtcc
     2161 gcgcgatggc acccctgtgc tgttcctgca cggtaacccg acctcctcct acgtgtggcg
     2221 caacatcatc ccgcatgttg caccgaccca tcgctgcatt gctccagacc tgatcggtat
     2281 gggcaaatcc gacaaaccag acctgggtta tttcttcgac gaccacgtcc gcttcatgga
     2341 tgccttcatc gaagccctgg gtctggaaga ggtcgtcctg gtcattcacg actggggctc
     2401 cgctctgggt ttccactggg ccaagcgcaa tccagagcgc gtcaaaggta ttgcatttat
     2461 ggagttcatc cgccctatcc cgacctggga cgaatgggga ggttctatgg tgagcaaggg
     2521 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     2581 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     2641 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     2701 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     2761 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     2821 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     2881 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     2941 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     3001 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     3061 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     3121 gtccaaactg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     3181 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaatcta gagggcccgt
     3241 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     3301 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     3361 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     3421 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     3481 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     3541 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3601 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3661 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3721 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct
     3781 attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact
     3841 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat
     3901 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     3961 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca
     4021 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
     4081 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     4141 cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga
     4201 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     4261 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     4321 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     4381 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     4441 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     4501 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     4561 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     4621 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     4681 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     4741 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     4801 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg
     4861 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     4921 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     4981 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     5041 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5101 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5161 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5221 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5281 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5341 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5401 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5461 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5521 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5581 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5641 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5701 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5761 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5821 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5881 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5941 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6001 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6061 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6121 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6181 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6241 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6301 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6361 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6421 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6481 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6541 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6601 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6661 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6721 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6781 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6841 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6901 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6961 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7021 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7081 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7141 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7201 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7261 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
