LOCUS       sequence-389873-        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-165-166
FEATURES             Location/Qualifiers
     source          1..5687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        40..324
                     /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     247..266
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             370..387
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             391..423
                     /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(427..435,436..450)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1170..1191
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1297..1318)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1558..1577)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2127..2144)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2297..2383
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2475..2502
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2521..2612
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2613..2681,2682..3473)
                     /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(2831..2850)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3644..4232
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4133..4152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4386..4403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4418..4558
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4484..4503)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4644..4666
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(4822..5687,1..13))
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat tgtccatatt
       61 gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc aaaccggtaa
      121 ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta
      181 acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac ggcgtcacac
      241 tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga cgctttttat
      301 cgcaactctc tactgtttct ccatacccgt tttttgggct aacaggagga attaaccatg
      361 gggggttctc atcatcatca tcatcatggt atggctagca tgactggtgg acagcaaatg
      421 ggtcgggatc tgtacgacga tgacgataag gatccgagct cgagaatggc agaaatcggt
      481 actggctttc cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat gcactacgtc
      541 gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc gacctcctcc
      601 tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat tgctccagac
      661 ctgatcggta tgggcaaatc cgacaaacca gacctgggtt atttcttcga cgaccacgtc
      721 cgcttcatgg atgccttcat cgaagccctg ggtctggaag aggtcgtcct ggtcattcac
      781 gactggggct ccgctctggg tttccactgg gccaagcgca atccagagcg cgtcaaaggt
      841 attgcattta tggagttcat ccgccctatc ccgacctggg acgaatggcc agaatttgcc
      901 cgcgagacct tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat catcgatggt
      961 gaaaacgtct atatcaaggc cgacaagcag aagaacggca tcaaggcgaa cttcaagatc
     1021 cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc actaccagca gaacaccccc
     1081 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcgtcca gtccaaactt
     1141 tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1201 gggatcactc tcggcatgga cgagctgtac aagggcggta ccggagggag catggtgagc
     1261 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1321 aacggccaca agttcagcgt gtccggcgag ggtgagggcg atgccaccta cggcaagctg
     1381 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1441 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1501 ttcttcaagt ccgccatgcc cgaaggctac atccaggagc gcaccatctt cttcaaggac
     1561 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1621 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca taagctggag
     1681 tacaaccttg gcgtttttat cgagggtacg ctgccgatgg gtgtcgtccg cccgctgact
     1741 gaagtcgaga tggaccatta ccgcgagccg ttcctgaatc ctgttgaccg cgagccactg
     1801 tggcgcttcc caaacgagct gccaatcgcc ggtgagccag cgaacatcgt cgcgctggtc
     1861 gaagaataca tggactggct gcaccagtcc cctgtcccga agctgctgtt ctggggcacc
     1921 ccaggcgttc tgatcccacc ggccgaagcc gctcgcctgg ccaaaagcct gcctaactgc
     1981 aaggctgtgg acatcggccc gggtctgaat ctgctgcaag aagacaaccc ggacctgatc
     2041 ggcagcgaga tcgcgcgctg gctgtcgacg ctcgagattt ccggctgaaa gcttggctgt
     2101 tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt
     2161 ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     2221 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta
     2281 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     2341 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt
     2401 gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag
     2461 gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct
     2521 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2581 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     2641 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2701 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2761 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2821 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     2881 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2941 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     3001 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     3061 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3121 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     3181 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3241 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3301 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     3361 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3421 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3481 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3541 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3601 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3661 tgcgcgtaat ctgctgcttg caaacaaaac aaccaccgct accagcggtg gtttgtttgc
     3721 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3781 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3841 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3901 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3961 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4021 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4081 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4141 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4201 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4261 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4321 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4381 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4441 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg
     4501 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     4561 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4621 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4681 accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc atgcataatg
     4741 tgcctgtcaa atggacgaag cagggattct gcaaacccta tgctactccg tcaagccgtc
     4801 aattgtctga ttcgttacca attatgacaa cttgacggct acatcattca ctttttcttc
     4861 acaaccggca cggaactcgc tcgggctggc cccggtgcat tttttaaata cccgcgagaa
     4921 atagagttga tcgtcaaaac caacattgcg accgacggtg gcgataggca tccgggtggt
     4981 gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg cgccagctta agacgctaat
     5041 ccctaactgc tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa catgctgtgc
     5101 gacgctggcg atatcaaaat tgctgtctgc caggtgatcg ctgatgtact gacaagcctc
     5161 gcgtacccga ttatccatcg gtggatggag cgactcgtta atcgcttcca tgcgccgcag
     5221 taacaattgc tcaagcagat ttatcgccag cagctccgaa tagcgccctt ccccttgccc
     5281 ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt catccgggcg
     5341 aaagaacccc gtattggcaa atattgacgg ccagttaagc cattcatgcc agtaggcgcg
     5401 cggacgaaag taaacccact ggtgatacca ttcgcgagcc tccggatgac gaccgtagtg
     5461 atgaatctct cctggcggga acagcaaaat atcacccggt cggcaaacaa attctcgtcc
     5521 ctgatttttc accaccccct gaccgcgaat ggtgagattg agaatataac ctttcattcc
     5581 cagcggtcgg tcgataaaaa aatcgagata accgttggcc tcaatcggcg ttaaacccgc
     5641 caccagatgg gcattaaacg agtatcccgg cagcagggga tcatttt
//
