LOCUS       sequence-389852-        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-Na0.5
FEATURES             Location/Qualifiers
     source          1..5687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        13..297
                     /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     220..239
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             343..360
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             364..396
                     /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(400..408,409..423)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1077..1098
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1204..1225)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2100..2117)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2270..2356
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2448..2475
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2494..2585
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2586..2654,2655..3446)
                     /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(2804..2823)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3617..4205
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4106..4125
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4359..4376
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4391..4531
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4457..4476)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4617..4639
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4795..5673)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 cgccattcag agaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct
       61 tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac
      121 aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa
      181 aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca
      241 taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc
      301 cgttttttgg gctaacagga ggaattaacc atggggggtt ctcatcatca tcatcatcat
      361 ggtatggcta gcatgactgg tggacagcaa atgggtcggg atctgtacga cgatgacgat
      421 aaggatccga gctcgagaat gacagaaatc ggtactggct ttccattcga cccccattat
      481 gtggaagtcc tgggcgcgcg catgcactac gtcgatgttg gtccgcgcga tggcatccct
      541 gtgctgttcc tgcatggtaa cccaacctcc tcctacgtgt ggcgcaacat catcccgcat
      601 gtcgcaccga cccatcgctg cattgctcca gacctgatcg gtatgggcaa atccgacaaa
      661 ccagacctgg gttatttctt cgacgaccac gtccgcttca tggatgcctt catcgaagcc
      721 ctgggtctgg aagaggtcgt cctggtcatt cacgactggg gctccgctct gggtttccac
      781 tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt catccgccct
      841 atcccgacct gggacgaatg gccgggggag aacgtctata tcaaggccga caagcagaag
      901 aacggcatca aggcgaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc
      961 gcctaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1021 cactccctga gcgtccagtc caaactttcg aaagacccca atgagaagcg cgatcacatg
     1081 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     1141 ggcggtaccg gagggagcat ggtgagcaag ggcggggagc tgttcaccgg ggtggtgccc
     1201 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggt
     1261 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1321 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     1381 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacatc
     1441 caggagcgca ccatcctctt caagggcgac ggcaactaca agacccgcgc cgaggtgaag
     1501 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcgtcgactt caaggaggac
     1561 ggcaacatcc tggggcataa gctggagtac aaccttggcg cccgcgggac cttccaggcc
     1621 ttccgcacca ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt tatcgagggt
     1681 acgctgccga tgggtgtcgt ccgcctgctg actgaagtcg agatggacca ttaccgcgag
     1741 ccgttcctga atcctgttga ccgcgagcca ctgtggcgct tcccaaacga gctgccaatc
     1801 gccggtgagc cagcgaacat cgtcgcgctg gtcgaagaat acatggactg gctgcaccag
     1861 tcccctgtcc cgaagctgct gttctggggc accccaggcg ttctgatccc accggccgaa
     1921 gccgctcgcc tggccaaaag cctgcctaac tgcaaggctg tggacatcgg cccgggtctg
     1981 aatctgctgc aagaagacaa cccggacctg atcggcagcg agatcgcgcg ctggctgtcg
     2041 acgctcggga tttccggctg aaagcttggc tgttttggcg gatgagagaa gattttcagc
     2101 ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc
     2161 agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc
     2221 gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg
     2281 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     2341 cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg
     2401 gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct
     2461 gacggatggc ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa
     2521 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2581 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     2641 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     2701 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     2761 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     2821 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2881 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2941 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     3001 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     3061 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     3121 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3181 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3241 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     3301 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     3361 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     3421 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     3481 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     3541 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3601 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3661 aacaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3721 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3781 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3841 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3901 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3961 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4021 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4081 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4141 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4201 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4261 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4321 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4381 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4441 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     4501 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     4561 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     4621 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa
     4681 ttcgcgcgcg aaggcgaagc ggcatgcata atgtgcctgt caaatggacg aagcagggat
     4741 tctgcaaacc ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga
     4801 caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct
     4861 ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt
     4921 gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat
     4981 acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga
     5041 cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc
     5101 tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg
     5161 gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc
     5221 cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc
     5281 gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga
     5341 cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata
     5401 ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa
     5461 aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg
     5521 aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag
     5581 ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc
     5641 cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcc
//
