LOCUS       sequence-389868-        5687 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Green fluorescent Ca2+ ion indicator for bacterial expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5687)
  AUTHORS   Zhu W, Takeuchi S, Imai S, Terada T, Ueda T, Nasu Y, Terai T, 
            Campbell RE
  TITLE     Chemigenetic indicators based on synthetic chelators and green 
            fluorescent protein.
  JOURNAL   Nat Chem Biol. 2023 Jan;19(1):38-44. doi: 
            10.1038/s41589-022-01134-z. Epub 2022 Sep 22.
  PUBMED    36138142
REFERENCE   2  (bases 1 to 5687)
  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: 
            "10.1038/s41589-022-01134-z"; journalName: "Nat Chem Biol"; date: 
            "2023-01"; volume: "19"; issue: "1"; pages: "38-44"
COMMENT     Sequence Label: pBad-HaloGFP-164-165
FEATURES             Location/Qualifiers
     source          1..5687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        20..304
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     227..246
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             350..367
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             371..403
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(407..415,416..430)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1147..1168
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1274..1295)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1535..1554)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2107..2124)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2277..2363
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2455..2482
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2501..2592
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2593..2661,2662..3453)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2811..2830)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3624..4212
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4113..4132
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4366..4383
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4398..4538
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4464..4483)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4624..4646
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4802..5680)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 atactcccgc cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac
       61 tgcgtctttt actggctctt ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt
      121 ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac
      181 ggcagaaaag tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt
      241 ttatccataa gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct
      301 ccatacccgt tttttgggct aacaggagga attaaccatg gggggttctc atcatcatca
      361 tcatcatggt atggctagca tgactggtgg acagcaaatg ggtcgggatc tgtacgacga
      421 tgacgataag gatccgagct cgagaatggc agaaatcggt actggctttc cattcgaccc
      481 ccattatgtg gaagtcctgg gcgagcgcat gcactacgtc gatgttggtc cgcgcgatgg
      541 cacccctgtg ctgttcctgc acggtaaccc gacctcctcc tacgtgtggc gcaacatcat
      601 cccgcatgtt gcaccgaccc atcgctgcat tgctccagac ctgatcggta tgggcaaatc
      661 cgacaaacca gacctgggtt atttcttcga cgaccacgtc cgcttcatgg atgccttcat
      721 cgaagccctg ggtctggaag aggtcgtcct ggtcattcac gactggggct ccgctctggg
      781 tttccactgg gccaagcgca atccagagcg cgtcaaaggt attgcattta tggagttcat
      841 ccgccctatc ccgacctggg acgaatggcc agaatttgcc cgcgagacct tccaggcctt
      901 ccgcaccacc gacgtcggcc gcaagctgat catcggtgaa aacgtctata tcaaggccga
      961 caagcagaag aacggcatca aggcgaactt caagatccgc cacaacatcg aggacggcgg
     1021 cgtgcagctc gcctaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1081 gcccgacaac cactacctga gcgtccagtc caaactttcg aaagacccca acgagaagcg
     1141 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     1201 gctgtacaag ggcggtaccg gagggagcat ggtgagcaag ggcgaggagc tgttcaccgg
     1261 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     1321 cggcgagggt gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     1381 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     1441 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     1501 aggctacatc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     1561 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     1621 caaggaggac ggcaacatcc tggggcataa gctggagtac aaccttggca acgtttttat
     1681 cgagggtacg ctgccgatgg gtgtcgtccg cccgctgact gaagtcgaga tggaccatta
     1741 ccgcgagccg ttcctgaatc ctgttgaccg cgagccactg tggcgcttcc caaacgagct
     1801 gccaatcgcc ggtgagccag cgaacatcgt cgcgctggtc gaagaataca tggactggct
     1861 gcaccagtcc cctgtcccga agctgctgtt ctggggcacc ccaggcgttc tgatcccacc
     1921 ggccgaagcc gctcgcctgg ccaaaagcct gcctaactgc aaggctgtgg acatcggccc
     1981 gggtctgaat ctgctgcaag aagacaaccc ggacctgatc ggcagcgaga tcgcgcgctg
     2041 gctgtcgacg ctcgagattt ccggctgaaa gcttggctgt tttggcggat gagagaagat
     2101 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc
     2161 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg
     2221 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa
     2281 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
     2341 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc
     2401 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg
     2461 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata
     2521 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2581 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     2641 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     2701 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     2761 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     2821 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     2881 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     2941 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     3001 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     3061 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     3121 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     3181 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     3241 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     3301 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     3361 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     3421 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     3481 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3541 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3601 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3661 caaacaaaac aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3721 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     3781 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3841 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3901 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3961 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4021 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4081 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4141 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4201 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4261 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4321 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4381 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4441 caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     4501 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4561 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4621 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4681 agatcaattc gcgcgcgaag gcgaagcggc atgcataatg tgcctgtcaa atggacgaag
     4741 cagggattct gcaaacccta tgctactccg tcaagccgtc aattgtctga ttcgttacca
     4801 attatgacaa cttgacggct acatcattca ctttttcttc acaaccggca cggaactcgc
     4861 tcgggctggc cccggtgcat tttttaaata cccgcgagaa atagagttga tcgtcaaaac
     4921 caacattgcg accgacggtg gcgataggca tccgggtggt gctcaaaagc agcttcgcct
     4981 ggctgatacg ttggtcctcg cgccagctta agacgctaat ccctaactgc tggcggaaaa
     5041 gatgtgacag acgcgacggc gacaagcaaa catgctgtgc gacgctggcg atatcaaaat
     5101 tgctgtctgc caggtgatcg ctgatgtact gacaagcctc gcgtacccga ttatccatcg
     5161 gtggatggag cgactcgtta atcgcttcca tgcgccgcag taacaattgc tcaagcagat
     5221 ttatcgccag cagctccgaa tagcgccctt ccccttgccc ggcgttaatg atttgcccaa
     5281 acaggtcgct gaaatgcggc tggtgcgctt catccgggcg aaagaacccc gtattggcaa
     5341 atattgacgg ccagttaagc cattcatgcc agtaggcgcg cggacgaaag taaacccact
     5401 ggtgatacca ttcgcgagcc tccggatgac gaccgtagtg atgaatctct cctggcggga
     5461 acagcaaaat atcacccggt cggcaaacaa attctcgtcc ctgatttttc accaccccct
     5521 gaccgcgaat ggtgagattg agaatataac ctttcattcc cagcggtcgg tcgataaaaa
     5581 aatcgagata accgttggcc tcaatcggcg ttaaacccgc caccagatgg gcattaaacg
     5641 agtatcccgg cagcagggga tcattttgcg cttcagccat acttttc
//
