LOCUS       sequence-388465-        6739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein production of the split HaloTag based calcium recorder 
            Caprola_15 in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6739)
  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 6739)
  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: pET-51b(+)-Caprola_15
FEATURES             Location/Qualifiers
     source          1..6739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     54..71
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    86..225
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(151..170)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     311..333
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(327..518)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1090..2172)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYITRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2134..2153
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2173..2250)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2456..2475
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2563..2582
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2563..2581
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2582..2606
                     /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)."
     RBS             2621..2643
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2659..2682
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2689..2703
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             2758..3198
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAEL
                     RHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVVMMTAK"
     CDS             4297..4326
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(4349..4367)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4363..4410
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4447..4902
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4534..4553)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4744..4765
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5017..5808,5809..5877))
                     /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     5640..5659
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5878..5970)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6051..6639
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6540..6559
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
       61 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
      121 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
      181 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
      241 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
      301 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
      361 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
      421 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
      481 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
      541 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
      601 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
      661 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
      721 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
      781 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
      841 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
      901 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
      961 aggagcacga tcatgctagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     1021 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     1081 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     1141 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     1201 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     1261 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     1321 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc
     1381 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     1441 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     1501 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgat
     1561 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     1621 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     1681 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     1741 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     1801 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     1861 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     1921 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     1981 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     2041 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     2101 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     2161 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     2221 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     2281 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     2341 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     2401 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     2461 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     2521 gtccggcgta gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactatag
     2581 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
     2641 agatatacca tggcaagctg gagccacccg cagttcgaaa agggtgcaga tgacgacgac
     2701 aaggtaccgc atggcggttc tgcccgcgag accttccagg ccttccgcac cggttctgac
     2761 caactgactg aagagcagat cgcagaattt aaagaggctt tctccctatt tgacaaggac
     2821 ggggatggga caataacaac caaggagctg gggacggtga tgcggtctct ggggcagaac
     2881 cccacagaag cagagctgca ggacatgatc aatgaagtag atgccgacgg tgacggcaca
     2941 atcgacttcc ctgagttcct gacaatgatg gcaagaaaaa tgaaagacac agacagtgaa
     3001 gaagaaatta gagaagcgtt ccgtgtgttt gataaggatg gcaatggcta catcagtgca
     3061 gcagagcttc gccacgtgat gacaaacctt ggagagaagt taacagatga agaggttgat
     3121 gaaatgatca gggaagcaga catcgatggg gatggtcagg taaactacga agagtttgta
     3181 gtgatgatga cagcgaagga attccctcct cctccgccac cgccaccacc gccgccgccg
     3241 ccaccaccac ctcctccgcc tcctccacca ccaccgccac cgcctccacc acctggcggc
     3301 agcagggtcg actcatcacg tcgtaagttc aataaggacg gtcacgcact gagagctata
     3361 ggtcggctga gctcactcga gggcggttct gacgtcggcc gcaagctgat catcgatcag
     3421 aacgttttta tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac tgaagtcgag
     3481 atggaccatt accgcgagcc gttcctgaat cctgttgacc gcgagccact gtggcgcttc
     3541 ccaaacgagc tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt cgaagaatac
     3601 atggactggc tgcaccagtc ccctgtcccg aagctgctgt tctggggcac cccaggcgtt
     3661 ctgatcccac cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg caaggctgtg
     3721 gacatcggcc cgggtctgaa tctgctgcaa gaagacaacc cggacctgat cggcagcgag
     3781 atcgcgcgct ggctgtctac tctggagatt ggtggcaccg gtggctctgg cggtaccggt
     3841 ggctctggcg gttctatcgg tactggcttt ccattcgacc cccattatgt ggaagtcctg
     3901 ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg
     3961 cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca tcccacatgt tgcaccgacc
     4021 catcgctgca ttgctccaga cctgatcggt atgggcaaat ccgacaaacc agacctgggt
     4081 tatttcttcg acgaccacgt ccgcttcatg gatgccttca tcgaagccct gggtctggaa
     4141 gaggtcgtcc tggtcattca cgactggggc tccgctctgg gtttccactg ggccaagcgc
     4201 aatccagagc gcgtcaaagg tattgcattt atggagttca tccgccctat cccgacctgg
     4261 gacgaatggg ctccgggctt ctcctcaatt tccgctcatc accaccatca tcaccatcac
     4321 caccactaat taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg
     4381 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     4441 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4501 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4561 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4621 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4681 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4741 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4801 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4861 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat
     4921 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4981 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5041 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5101 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5161 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5221 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5281 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5341 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5401 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5461 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5521 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5581 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacgtga
     5641 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5701 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5761 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5821 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5881 cttccttttt caatcatgat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5941 tatttgaatg tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag
     6001 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6061 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6121 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6181 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     6241 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6301 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6361 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6421 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6481 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6541 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6601 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6661 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6721 gattctgtgg ataaccgta
//
