LOCUS       sequence-389864-        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-175-176
FEATURES             Location/Qualifiers
     source          1..5687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        28..312
                     /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     235..254
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             358..375
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             379..411
                     /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(415..423,424..438)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1188..1209
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1315..1336)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1576..1595)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2115..2132)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2285..2371
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2463..2490
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2509..2600
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2601..2669,2670..3461)
                     /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(2819..2838)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3632..4220
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4121..4140
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4374..4391
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4406..4546
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4472..4491)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4632..4654
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(4810..5687,1))
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 tacttttcat actcccgcca ttcagagaag aaaccaattg tccatattgc atcagacatt
       61 gccgtcactg cgtcttttac tggctcttct cgctaaccaa accggtaacc ccgcttatta
      121 aaagcattct gtaacaaagc gggaccaaag ccatgacaaa aacgcgtaac aaaagtgtct
      181 ataatcacgg cagaaaagtc cacattgatt atttgcacgg cgtcacactt tgctatgcca
      241 tagcattttt atccataaga ttagcggatc ctacctgacg ctttttatcg caactctcta
      301 ctgtttctcc atacccgttt tttgggctaa caggaggaat taaccatggg gggttctcat
      361 catcatcatc atcatggtat ggctagcatg actggtggac agcaaatggg tcgggatctg
      421 tacgacgatg acgataagga tccgagctcg agaatggcag aaatcggtac tggctttcca
      481 ttcgaccccc attatgtgga agtcctgggc gagcgcatgc actacgtcga tgttggtccg
      541 cgcgatggca cccctgtgct gttcctgcac ggtaacccga cctcctccta cgtgtggcgc
      601 aacatcatcc cgcatgttgc accgacccat cgctgcattg ctccagacct gatcggtatg
      661 ggcaaatccg acaaaccaga cctgggttat ttcttcgacg accacgtccg cttcatggat
      721 gccttcatcg aagccctggg tctggaagag gtcgtcctgg tcattcacga ctggggctcc
      781 gctctgggtt tccactgggc caagcgcaat ccagagcgcg tcaaaggtat tgcatttatg
      841 gagttcatcc gccctatccc gacctgggac gaatggccag aatttgcccg cgagaccttc
      901 caggccttcc gcaccaccga cgtcggccgc aagctgatca tcgatcagaa cgtttttatc
      961 gagggtacgc tgccgggtga aaacgtctat atcaaggccg acaagcagaa gaacggcatc
     1021 aaggcgaact tcaagatccg ccacaacatc gaggacggcg gcgtgcagct cgcctaccac
     1081 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1141 agcgtccagt ccaaactttc gaaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1201 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggcggtacc
     1261 ggagggagca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1321 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg tgagggcgat
     1381 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1441 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     1501 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacat ccaggagcgc
     1561 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1621 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1681 ctggggcata agctggagta caaccttggc gtcgtccgcc cgctgactga agtcgagatg
     1741 gaccattacc gcgagccgtt cctgaatcct gttgaccgcg agccactgtg gcgcttccca
     1801 aacgagctgc caatcgccgg tgagccagcg aacatcgtcg cgctggtcga agaatacatg
     1861 gactggctgc accagtcccc tgtcccgaag ctgctgttct ggggcacccc aggcgttctg
     1921 atcccaccgg ccgaagccgc tcgcctggcc aaaagcctgc ctaactgcaa ggctgtggac
     1981 atcggcccgg gtctgaatct gctgcaagaa gacaacccgg acctgatcgg cagcgagatc
     2041 gcgcgctggc tgtcgacgct cgagatttcc ggctgaaagc ttggctgttt tggcggatga
     2101 gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag
     2161 aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg
     2221 aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag
     2281 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     2341 gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa
     2401 gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa
     2461 gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt tgtttatttt
     2521 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2581 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2641 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2701 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     2761 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2821 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     2881 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2941 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     3001 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     3061 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3121 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     3181 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     3241 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     3301 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     3361 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3421 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3481 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3541 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3601 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3661 gctgcttgca aacaaaacaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3721 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     3781 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3841 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3901 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3961 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4021 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4081 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4141 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4201 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4261 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4321 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4381 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4441 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4501 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     4561 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4621 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4681 gaggcagcag atcaattcgc gcgcgaaggc gaagcggcat gcataatgtg cctgtcaaat
     4741 ggacgaagca gggattctgc aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt
     4801 cgttaccaat tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg
     4861 gaactcgctc gggctggccc cggtgcattt tttaaatacc cgcgagaaat agagttgatc
     4921 gtcaaaacca acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag
     4981 cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg
     5041 gcggaaaaga tgtgacagac gcgacggcga caagcaaaca tgctgtgcga cgctggcgat
     5101 atcaaaattg ctgtctgcca ggtgatcgct gatgtactga caagcctcgc gtacccgatt
     5161 atccatcggt ggatggagcg actcgttaat cgcttccatg cgccgcagta acaattgctc
     5221 aagcagattt atcgccagca gctccgaata gcgcccttcc ccttgcccgg cgttaatgat
     5281 ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt
     5341 attggcaaat attgacggcc agttaagcca ttcatgccag taggcgcgcg gacgaaagta
     5401 aacccactgg tgataccatt cgcgagcctc cggatgacga ccgtagtgat gaatctctcc
     5461 tggcgggaac agcaaaatat cacccggtcg gcaaacaaat tctcgtccct gatttttcac
     5521 caccccctga ccgcgaatgg tgagattgag aatataacct ttcattccca gcggtcggtc
     5581 gataaaaaaa tcgagataac cgttggcctc aatcggcgtt aaacccgcca ccagatgggc
     5641 attaaacgag tatcccggca gcaggggatc attttgcgct tcagcca
//
