LOCUS       sequence-388477-        6739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein production of the split HaloTag based calcium recorder 
            Caprola_10 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_10
FEATURES             Location/Qualifiers
     source          1..6739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     207..226
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     314..333
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        314..332
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    333..357
                     /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             372..394
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             410..433
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             440..454
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             509..949
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAEL
                     RHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVVMMTAK"
     CDS             2048..2077
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(2100..2118)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2114..2161
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2198..2653
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2285..2304)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2495..2516
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2768..3559,3560..3628))
                     /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     3391..3410
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3629..3721)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3802..4390
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4291..4310
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4544..4561
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4576..4715
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4641..4660)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4801..4823
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4817..5008)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5580..6662)
                     /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     6624..6643
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(6663..6739,1))
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
       61 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
      121 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
      181 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
      241 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatcgatctc
      301 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
      361 ctagaaataa ttttgtttaa ctttaagaag gagatatacc atggcaagct ggagccaccc
      421 gcagttcgaa aagggtgcag atgacgacga caaggtaccg catggcggtt ctgcccgcga
      481 gaccttccag gccttccgca ccggttctga ccaactgact gaagagcaga tcgcagaatt
      541 taaagaggct ttctccctat ttgacaagga cggggatggg acaataacaa ccaaggagct
      601 ggggacggtg atgcggtctc tggggcagaa ccccacagaa gcagagctgc aggacatgat
      661 caatgaagta gatgccgacg gtgacggcac aatcgacttc cctgagttcc tgacaatgat
      721 ggcaagaaaa atgaaagaca cagacagtga agaagaaatt agagaagcgt tccgtgtgtt
      781 tgataaggat ggcaatggct acatcagtgc agcagagctt cgccacgtga tgacaaacct
      841 tggagagaag ttaacagatg aagaggttga tgaaatgatc agggaagcag acatcgatgg
      901 ggatggtcag gtaaactacg aagagtttgt agtgatgatg acagcgaagg aattccctcc
      961 tcctccgcca ccgccaccac cgccgccgcc gccaccacca cctcctccgc ctcctccacc
     1021 accaccgcca ccgcctccac cacctggcgg cagcagggtc gactcatcac gtcgtaaggt
     1081 caataagaca ggtcacgcac tgagagctat aggtcggctg agctcactcg agggcggttc
     1141 tgacgtcggc cgcaagctga tcatcgatca gaacgttttt atcgagggta cgctgccgat
     1201 gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat taccgcgagc cgttcctgaa
     1261 tcctgttgac cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg ccggtgagcc
     1321 agcgaacatc gtcgcgctgg tcgaagaata catggactgg ctgcaccagt cccctgtccc
     1381 gaagctgctg ttctggggca ccccaggcgt tctgatccca ccggccgaag ccgctcgcct
     1441 ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc ccgggtctga atctgctgca
     1501 agaagacaac ccggacctga tcggcagcga gatcgcgcgc tggctgtcta ctctggagat
     1561 tggtggcacc ggtggctctg gcggtaccgg tggctctggc ggttctatcg gtactggctt
     1621 tccattcgac ccccattatg tggaagtcct gggcgagcgc atgcactacg tcgatgttgg
     1681 tccgcgcgat ggcacccctg tgctgttcct gcacggtaac ccgacctcct cctacgtgtg
     1741 gcgcaacatc atcccacatg ttgcaccgac ccatcgctgc attgctccag acctgatcgg
     1801 tatgggcaaa tccgacaaac cagacctggg ttatttcttc gacgaccacg tccgcttcat
     1861 ggatgccttc atcgaagccc tgggtctgga agaggtcgtc ctggtcattc acgactgggg
     1921 ctccgctctg ggtttccact gggccaagcg caatccagag cgcgtcaaag gtattgcatt
     1981 tatggagttc atccgcccta tcccgacctg ggacgaatgg gctccgggct tctcctcaat
     2041 ttccgctcat caccaccatc atcaccatca ccaccactaa ttaacctagg ctgctgccac
     2101 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     2161 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     2221 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2281 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2341 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2401 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2461 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2521 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2581 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     2641 acgtttacaa tttctggcgg cacgatggca tgagattatc aaaaaggatc ttcacctaga
     2701 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2761 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2821 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2881 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     2941 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3001 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3061 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3121 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3181 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3241 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3301 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3361 cgagttgctc ttgcccggcg tcaatacgtg ataataccgc gccacatagc agaactttaa
     3421 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3481 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3541 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3601 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatcatga ttgaagcatt
     3661 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3721 ataggtcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3781 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3841 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3901 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3961 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4021 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4081 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4141 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4201 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4261 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4321 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4381 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4441 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4501 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4561 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4621 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     4681 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     4741 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4801 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     4861 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     4921 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     4981 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     5041 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     5101 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     5161 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     5221 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     5281 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     5341 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     5401 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     5461 tcatgctagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     5521 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     5581 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5641 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     5701 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     5761 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     5821 catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc
     5881 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     5941 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     6001 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgat atatttatgc
     6061 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     6121 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     6181 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     6241 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     6301 ccactgacgc gttgcgcgag aagattgtgt accgccgctt tacaggcttc gacgccgctt
     6361 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     6421 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     6481 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     6541 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     6601 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     6661 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     6721 ttgcgccatt cgatggtgt
//
