LOCUS       sequence-389844-        5663 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 5663)
  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 5663)
  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-155-164
FEATURES             Location/Qualifiers
     source          1..5663
                     /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     1127..1148
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1254..1275)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1515..1534)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2090..2107)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2260..2346
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2438..2465
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2484..2575
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2576..2644,2645..3436)
                     /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(2794..2813)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3607..4195
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4096..4115
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4349..4366
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4381..4521
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4447..4466)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4607..4629
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4785..5663)
                     /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 tcgtcttggt cattcacgac tggggctccg
      781 ctctgggttt ccactgggcc aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg
      841 agttcatccg ccctatcccg acctgggacg aatggccaga atttgcccgc gagaccttcc
      901 aggccttccg caccggtgaa aacgtctata tcaaggccga caagcagaag aacggcatca
      961 aggcgaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc gcctaccact
     1021 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     1081 gcgtccagtc caaactttcg aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     1141 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag ggcggtaccg
     1201 gagggagcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1261 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg
     1321 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     1381 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     1441 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacatc caggagcgca
     1501 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1561 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1621 tggggcataa gctggagtac aaccttggcc agaacgtttt tatcgagggt acgctgccga
     1681 tgggtgtcgt ccgcccgctg actgaagtcg agatggacca ttaccgcgag ccgttcctga
     1741 atcctgttga ccgcgagcca ctgtggcgct tcccaaacga gctgccaatc gccggtgagc
     1801 cagcgaacat cgtcgcgctg gtcgaagaat acatggactg gctgcaccag tcccctgtcc
     1861 cgaagctgct gttctggggc accccaggcg ttctgatccc accggccgaa gccgctcgcc
     1921 tggccaaaag cctgcctaac tgcaaggctg tggacatcgg cccgggtctg aatctgctgc
     1981 aagaagacaa cccggacctg atcggcagcg agatcgcgcg ctggctgtcg acgctcgaga
     2041 tttccggctg aaagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga
     2101 ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg
     2161 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg
     2221 tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
     2281 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     2341 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     2401 ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc
     2461 ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc
     2521 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2581 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2641 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2701 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2761 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     2821 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2881 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2941 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     3001 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     3061 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     3121 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3181 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3241 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3301 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3361 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3421 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3481 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3541 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3601 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aacaaccacc
     3661 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3721 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3781 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3841 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3901 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3961 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4021 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4081 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4141 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4201 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4261 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4321 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4381 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
     4441 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     4501 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4561 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4621 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg
     4681 aaggcgaagc ggcatgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc
     4741 ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg
     4801 gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg
     4861 cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg
     4921 gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc
     4981 tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac
     5041 ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga
     5101 tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg
     5161 ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc
     5221 gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc
     5281 ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta
     5341 agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga
     5401 gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc
     5461 ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga
     5521 ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg
     5581 gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg
     5641 ggatcatttt gcgcttcagc cat
//
