LOCUS       sequence-389876-        5657 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 5657)
  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 5657)
  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-143-154
FEATURES             Location/Qualifiers
     source          1..5657
                     /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(1465..1484)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2070..2087)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2240..2326
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2418..2445
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2464..2555
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2556..2624,2625..3416)
                     /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(2774..2793)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3587..4175
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4076..4095
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4329..4346
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4361..4501
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4427..4446)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4587..4609
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4765..5643)
                     /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 ggcagaaatc ggtactggct ttccattcga cccccattat
      481 gtggaagtcc tgggcgagcg catgcactac gtcgatgttg gtccgcgcga tggcacccct
      541 gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat catcccgcat
      601 gttgcaccga 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 gccaggtgaa aacgtctata tcaaggccga caagcagaag
      901 aacggcatca aggcgaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc
      961 gcctaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1021 cactacctga gcgtccagtc caaactttcg aaagacccca acgagaagcg cgatcacatg
     1081 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     1141 ggcggtaccg gagggagcat ggtgagcaag ggcgaggagc 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 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1501 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1561 ggcaacatcc tggggcataa gctggagtac aaccttggca ccgacgtcgg ccgcaagctg
     1621 atcatcgatc agaacgtttt tatcgagggt acgctgccga tgggtgtcgt ccgcccgctg
     1681 actgaagtcg agatggacca ttaccgcgag ccgttcctga atcctgttga ccgcgagcca
     1741 ctgtggcgct tcccaaacga gctgccaatc gccggtgagc cagcgaacat cgtcgcgctg
     1801 gtcgaagaat acatggactg gctgcaccag tcccctgtcc cgaagctgct gttctggggc
     1861 accccaggcg ttctgatccc accggccgaa gccgctcgcc tggccaaaag cctgcctaac
     1921 tgcaaggctg tggacatcgg cccgggtctg aatctgctgc aagaagacaa cccggacctg
     1981 atcggcagcg agatcgcgcg ctggctgtcg acgctcgaga tttccggctg aaagcttggc
     2041 tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga acgcagaagc
     2101 ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg
     2161 ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga
     2221 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     2281 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga
     2341 tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc
     2401 caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac
     2461 tcttttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2521 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     2581 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     2641 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     2701 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     2761 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     2821 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     2881 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     2941 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3001 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3061 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     3121 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3181 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3241 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     3301 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     3361 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     3421 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3481 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3541 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3601 ttctgcgcgt aatctgctgc ttgcaaacaa aacaaccacc gctaccagcg gtggtttgtt
     3661 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3721 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3781 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     3841 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3901 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3961 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4021 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4081 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4141 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4201 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4261 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4321 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     4381 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg
     4441 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     4501 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4561 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4621 atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg aaggcgaagc ggcatgcata
     4681 atgtgcctgt caaatggacg aagcagggat tctgcaaacc ctatgctact ccgtcaagcc
     4741 gtcaattgtc tgattcgtta ccaattatga caacttgacg gctacatcat tcactttttc
     4801 ttcacaaccg gcacggaact cgctcgggct ggccccggtg cattttttaa atacccgcga
     4861 gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggt
     4921 ggtgctcaaa agcagcttcg cctggctgat acgttggtcc tcgcgccagc ttaagacgct
     4981 aatccctaac tgctggcgga aaagatgtga cagacgcgac ggcgacaagc aaacatgctg
     5041 tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt actgacaagc
     5101 ctcgcgtacc cgattatcca tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg
     5161 cagtaacaat tgctcaagca gatttatcgc cagcagctcc gaatagcgcc cttccccttg
     5221 cccggcgtta atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg
     5281 gcgaaagaac cccgtattgg caaatattga cggccagtta agccattcat gccagtaggc
     5341 gcgcggacga aagtaaaccc actggtgata ccattcgcga gcctccggat gacgaccgta
     5401 gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg
     5461 tccctgattt ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat aacctttcat
     5521 tcccagcggt cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc
     5581 cgccaccaga tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagc
     5641 catacttttc atactcc
//
