LOCUS       Exported                4237 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Bioluminescent Reporter of Intracellular Calcium.
ACCESSION   .
VERSION     .
KEYWORDS    pT7-CalfluxVTN
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4237)
  AUTHORS   Yang J, Cumberbatch D, Centanni S, Shi SQ, Winder D, Webb D, Johnson
            CH
  TITLE     Coupling optogenetic stimulation with NanoLuc-based luminescence 
            (BRET) Ca++ sensing.
  JOURNAL   Nat Commun. 2016 Oct 27;7:13268. doi: 10.1038/ncomms13268.
  PUBMED    27786307
REFERENCE   2  (bases 1 to 4237)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4237
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             70..92
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             112..129
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             148..666
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIE"
     primer_bind     complement(193..214)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(454..473)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     801..822
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1069..1584
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(1638..1656)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1652..1699
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1796..2251
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1883..1902)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2093..2114
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2277..2381
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2382..3242
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2600..2619)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3413..4001
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3902..3921
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4155..4172
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
       61 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat
      121 catcatcatg gtatggctag cgaattcatg gtgagcaagg gcgaggagct gttcaccggg
      181 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
      241 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagctgat ctgcaccacc
      301 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgggctacgg cctgcagtgc
      361 ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
      421 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
      481 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
      541 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
      601 tatatcaccg ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac
      661 atcgaggacg gcggcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
      721 ggccccgtgc tgctgcccga caaccactac ctgagctacc agtccgccct gagcaaagac
      781 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc ccgcatgcaa
      841 gatgctagcg aagaagagct atcagagtgc tttcgcattt ttgacaagga tggaaatggt
      901 ttcatcgaca gggaggaatt tggagacatc atccgcctta ctggagaaca gctcacagat
      961 gaggatcccg atgagatatt tggagattca gacacagaca aaaatggaag gattgacttt
     1021 gatgagttcc tgaagatggt ggagaatgtc cagcctctgg ccgagctcat ggtcttcaca
     1081 ctcgaagatt tcgttgggga ctggcgacag acagccggct acaacctgga ccaagtcctt
     1141 gaacagggag gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac tccgatccaa
     1201 aggattgtcc tgagcggtga aaatgggctg aagatcgaca tccatgtcat catcccgtat
     1261 gaaggtctga gcggcgacca aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct
     1321 gtggatgatc atcactttaa ggtgatcctg cactatggca cactggtaat cgacggggtt
     1381 acgccgaaca tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc
     1441 aaaaagatca ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg
     1501 atcaaccccg acggctccct gctgttccga gtaaccatca acggagtgac cggctggcgg
     1561 ctgtgcgaac gcattctggc gtaagcttga tccggctgct aacaaagccc gaaaggaagc
     1621 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     1681 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatctggcg taatagcgaa
     1741 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     1801 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     1861 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     1921 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     1981 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2041 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2101 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2161 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2221 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     2281 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2341 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     2401 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     2461 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     2521 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     2581 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     2641 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2701 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2761 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2821 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2881 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     2941 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     3001 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     3061 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     3121 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     3181 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     3241 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     3301 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     3361 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3421 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3481 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3541 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3601 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3661 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3721 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3781 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3841 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3901 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3961 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4021 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4081 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     4141 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     4201 cgcctctccc cgcgcgttgg ccgattcatt aatgcag
//
