LOCUS       sequence-389843-        5681 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 5681)
  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 5681)
  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-143-146
FEATURES             Location/Qualifiers
     source          1..5681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        27..311
                     /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     234..253
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             357..374
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             378..410
                     /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(414..422,423..437)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1091..1112
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1218..1239)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1479..1498)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2108..2125)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2278..2364
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2456..2483
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2502..2593
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2594..2662,2663..3454)
                     /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(2812..2831)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3625..4213
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4114..4133
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4367..4384
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4399..4539
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4465..4484)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4625..4647
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4803..5681)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg
       61 ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa
      121 aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta
      181 taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat
      241 agcattttta tccataagat tagcggatcc tacctgacgc tttttatcgc aactctctac
      301 tgtttctcca tacccgtttt ttgggctaac aggaggaatt aaccatgggg ggttctcatc
      361 atcatcatca tcatggtatg gctagcatga ctggtggaca gcaaatgggt cgggatctgt
      421 acgacgatga cgataaggat ccgagctcga gaatggcaga aatcggtact ggctttccat
      481 tcgaccccca ttatgtggaa gtcctgggcg agcgcatgca ctacgtcgat gttggtccgc
      541 gcgatggcac ccctgtgctg ttcctgcacg gtaacccgac ctcctcctac gtgtggcgca
      601 acatcatccc gcatgttgca ccgacccatc gctgcattgc tccagacctg atcggtatgg
      661 gcaaatccga caaaccagac ctgggttatt tcttcgacga ccacgtccgc ttcatggatg
      721 ccttcatcga agccctgggt ctggaagagg tcgtcctggt cattcacgac tggggctccg
      781 ctctgggttt ccactgggcc aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg
      841 agttcatccg ccctatcccg acctgggacg aatggccagg tgaaaacgtc tatatcaagg
      901 ccgacaagca gaagaacggc atcaaggcga acttcaagat ccgccacaac atcgaggacg
      961 gcggcgtgca gctcgcctac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1021 tgctgcccga caaccactac ctgagcgtcc agtccaaact ttcgaaagac cccaacgaga
     1081 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1141 acgagctgta caagggcggt accggaggga gcatggtgag caagggcgag gagctgttca
     1201 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1261 tgtccggcga gggtgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1321 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     1381 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1441 ccgaaggcta catccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1501 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1561 acttcaagga ggacggcaac atcctggggc ataagctgga gtacaacctt ggcgagacct
     1621 tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat catcgatcag aacgttttta
     1681 tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac tgaagtcgag atggaccatt
     1741 accgcgagcc gttcctgaat cctgttgacc gcgagccact gtggcgcttc ccaaacgagc
     1801 tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt cgaagaatac atggactggc
     1861 tgcaccagtc ccctgtcccg aagctgctgt tctggggcac cccaggcgtt ctgatcccac
     1921 cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg caaggctgtg gacatcggcc
     1981 cgggtctgaa tctgctgcaa gaagacaacc cggacctgat cggcagcgag atcgcgcgct
     2041 ggctgtcgac gctcgagatt tccggctgaa agcttggctg ttttggcgga tgagagaaga
     2101 ttttcagcct gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc
     2161 ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc
     2221 gtagcgccga tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa
     2281 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     2341 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg
     2401 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag
     2461 gccatcctga cggatggcct ttttgcgttt ctacaaactc ttttgtttat ttttctaaat
     2521 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2581 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     2641 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     2701 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     2761 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     2821 cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc
     2881 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     2941 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3001 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3061 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3121 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3181 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     3241 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3301 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     3361 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3421 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     3481 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3541 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3601 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3661 gcaaacaaaa caaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3721 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3781 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3841 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3901 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3961 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4021 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4081 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4141 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4201 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4261 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4321 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4381 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4441 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     4501 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     4561 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4621 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     4681 cagatcaatt cgcgcgcgaa ggcgaagcgg catgcataat gtgcctgtca aatggacgaa
     4741 gcagggattc tgcaaaccct atgctactcc gtcaagccgt caattgtctg attcgttacc
     4801 aattatgaca acttgacggc tacatcattc actttttctt cacaaccggc acggaactcg
     4861 ctcgggctgg ccccggtgca ttttttaaat acccgcgaga aatagagttg atcgtcaaaa
     4921 ccaacattgc gaccgacggt ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc
     4981 tggctgatac gttggtcctc gcgccagctt aagacgctaa tccctaactg ctggcggaaa
     5041 agatgtgaca gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc gatatcaaaa
     5101 ttgctgtctg ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg attatccatc
     5161 ggtggatgga gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg ctcaagcaga
     5221 tttatcgcca gcagctccga atagcgccct tccccttgcc cggcgttaat gatttgccca
     5281 aacaggtcgc tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc cgtattggca
     5341 aatattgacg gccagttaag ccattcatgc cagtaggcgc gcggacgaaa gtaaacccac
     5401 tggtgatacc attcgcgagc ctccggatga cgaccgtagt gatgaatctc tcctggcggg
     5461 aacagcaaaa tatcacccgg tcggcaaaca aattctcgtc cctgattttt caccaccccc
     5521 tgaccgcgaa tggtgagatt gagaatataa cctttcattc ccagcggtcg gtcgataaaa
     5581 aaatcgagat aaccgttggc ctcaatcggc gttaaacccg ccaccagatg ggcattaaac
     5641 gagtatcccg gcagcagggg atcattttgc gcttcagcca t
//
