LOCUS       sequence-389865-        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-171-174
FEATURES             Location/Qualifiers
     source          1..5681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        14..298
                     /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     221..240
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             344..361
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             365..397
                     /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(401..409,410..424)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1162..1183
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1289..1310)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1550..1569)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2095..2112)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2265..2351
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2443..2470
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2489..2580
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2581..2649,2650..3441)
                     /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(2799..2818)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3612..4200
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4101..4120
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4354..4371
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4386..4526
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4452..4471)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4612..4634
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4790..5668)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 ccgccattca gagaagaaac caattgtcca tattgcatca gacattgccg tcactgcgtc
       61 ttttactggc tcttctcgct aaccaaaccg gtaaccccgc ttattaaaag cattctgtaa
      121 caaagcggga ccaaagccat gacaaaaacg cgtaacaaaa gtgtctataa tcacggcaga
      181 aaagtccaca ttgattattt gcacggcgtc acactttgct atgccatagc atttttatcc
      241 ataagattag cggatcctac ctgacgcttt ttatcgcaac tctctactgt ttctccatac
      301 ccgttttttg ggctaacagg aggaattaac catggggggt tctcatcatc atcatcatca
      361 tggtatggct agcatgactg gtggacagca aatgggtcgg gatctgtacg acgatgacga
      421 taaggatccg agctcgagaa tggcagaaat cggtactggc tttccattcg acccccatta
      481 tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc
      541 tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca tcatcccgca
      601 tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca aatccgacaa
      661 accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct tcatcgaagc
      721 cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc tgggtttcca
      781 ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt tcatccgccc
      841 tatcccgacc tgggacgaat ggccagaatt tgcccgcgag accttccagg ccttccgcac
      901 caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg gtgaaaacgt
      961 ctatatcaag gccgacaagc agaagaacgg catcaaggcg aacttcaaga tccgccacaa
     1021 catcgaggac ggcggcgtgc agctcgccta ccactaccag cagaacaccc ccatcggcga
     1081 cggccccgtg ctgctgcccg acaaccacta cctgagcgtc cagtccaaac tttcgaaaga
     1141 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     1201 tctcggcatg gacgagctgt acaagggcgg taccggaggg agcatggtga gcaagggcga
     1261 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     1321 caagttcagc gtgtccggcg agggtgaggg cgatgccacc tacggcaagc tgaccctgaa
     1381 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     1441 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     1501 gtccgccatg cccgaaggct acatccagga gcgcaccatc ttcttcaagg acgacggcaa
     1561 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     1621 gaagggcatc gacttcaagg aggacggcaa catcctgggg cataagctgg agtacaacct
     1681 tggcatgggt gtcgtccgcc cgctgactga agtcgagatg gaccattacc gcgagccgtt
     1741 cctgaatcct gttgaccgcg agccactgtg gcgcttccca aacgagctgc caatcgccgg
     1801 tgagccagcg aacatcgtcg cgctggtcga agaatacatg gactggctgc accagtcccc
     1861 tgtcccgaag ctgctgttct ggggcacccc aggcgttctg atcccaccgg ccgaagccgc
     1921 tcgcctggcc aaaagcctgc ctaactgcaa ggctgtggac atcggcccgg gtctgaatct
     1981 gctgcaagaa gacaacccgg acctgatcgg cagcgagatc gcgcgctggc tgtcgacgct
     2041 cgagatttcc ggctgaaagc ttggctgttt tggcggatga gagaagattt tcagcctgat
     2101 acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag
     2161 cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg
     2221 tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg
     2281 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga
     2341 gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc
     2401 gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg
     2461 atggcctttt tgcgtttcta caaactcttt tgtttatttt tctaaataca ttcaaatatg
     2521 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     2581 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     2641 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     2701 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     2761 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     2821 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     2881 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     2941 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3001 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3061 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3121 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3181 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3241 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     3301 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     3361 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     3421 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     3481 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     3541 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3601 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaacaa
     3661 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3721 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3781 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3841 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3901 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3961 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4021 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4081 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4141 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4201 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4261 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4321 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4381 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg
     4441 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     4501 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     4561 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4621 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc
     4681 gcgcgaaggc gaagcggcat gcataatgtg cctgtcaaat ggacgaagca gggattctgc
     4741 aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt cgttaccaat tatgacaact
     4801 tgacggctac atcattcact ttttcttcac aaccggcacg gaactcgctc gggctggccc
     4861 cggtgcattt tttaaatacc cgcgagaaat agagttgatc gtcaaaacca acattgcgac
     4921 cgacggtggc gataggcatc cgggtggtgc tcaaaagcag cttcgcctgg ctgatacgtt
     4981 ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg gcggaaaaga tgtgacagac
     5041 gcgacggcga caagcaaaca tgctgtgcga cgctggcgat atcaaaattg ctgtctgcca
     5101 ggtgatcgct gatgtactga caagcctcgc gtacccgatt atccatcggt ggatggagcg
     5161 actcgttaat cgcttccatg cgccgcagta acaattgctc aagcagattt atcgccagca
     5221 gctccgaata gcgcccttcc ccttgcccgg cgttaatgat ttgcccaaac aggtcgctga
     5281 aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt attggcaaat attgacggcc
     5341 agttaagcca ttcatgccag taggcgcgcg gacgaaagta aacccactgg tgataccatt
     5401 cgcgagcctc cggatgacga ccgtagtgat gaatctctcc tggcgggaac agcaaaatat
     5461 cacccggtcg gcaaacaaat tctcgtccct gatttttcac caccccctga ccgcgaatgg
     5521 tgagattgag aatataacct ttcattccca gcggtcggtc gataaaaaaa tcgagataac
     5581 cgttggcctc aatcggcgtt aaacccgcca ccagatgggc attaaacgag tatcccggca
     5641 gcaggggatc attttgcgct tcagccatac ttttcatact c
//
