LOCUS       Exported                7532 bp ds-DNA     circular SYN 27-JUL-2022
DEFINITION  pCAG-CalfluxCTN, BRET-based calcium sensor CalfluxCTN under CAG 
            promoter with puromycin selection marker for stable transfection in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7532)
  AUTHORS   Cumberbatch et al.
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Jul 27, 2022 from SnapGene Viewer 4.3.10
            https://www.snapgene.com
COMMENT     Used in Cumberbatch et al. (2022) A BRET Ca2+ sensor enables 
            high-throughput screening in the presence of background 
            fluorescence.
FEATURES             Location/Qualifiers
     source          1..7532
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        15..394
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        396..669
                     /label=chicken beta actin promoter
     intron          670..1686
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     regulatory      1845..1854
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1851..3299
                     /codon_start=1
                     /product="CalfluxCTN
                     BRET-based calcium sensor composed of the following 5 
                     segments:
                     Segment 1: Clover C?9 - Amino acid residues 1-230 of Clover
                     Segment 2: Linker - MQVAD
                     Segment 3: TnC - EF-hand motif derived from Opsanus tau 
                     troponin C
                     Segment 4: Linker - IYPEL
                     Segment 5: NanoLuc - NanoLuc luciferase"
                     /label=CalfluxCTN
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGIMQVADASEEELSECFRIFDFDGNGFIDREEFGDIIRLTGEQLTDEDVDEIF
                     GDSDTDKNGRIDFDEFLKMVENVQPIYPELMVFTLEDFVGDWRQTAGYNLDQVLEQGGV
                     SSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDH
                     HFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINP
                     DGSLLFRVTINGVTGWRLCERILA"
     polyA_signal    3399..3623
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3669..4097
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        4111..4440
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4291..4426
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4449..5048
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    5208..5329
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5378..5394)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5402..5418
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(5426..5456)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5471..5492
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5780..6365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6536..7396)
                     /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"
     promoter        complement(7397..7501)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
       61 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      121 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      181 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      241 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      301 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      361 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt
      421 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt
      481 attttgtgca gcgatggggg cggggggggg gggcgcgcgc caggcggggc ggggcggggc
      541 gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc
      601 cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg
      661 cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg
      721 cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc
      781 ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg
      841 ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg
      901 ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc
      961 tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc
     1021 gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg
     1081 cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc
     1141 cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt
     1201 acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg
     1261 ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg
     1321 agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc
     1381 gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc
     1441 cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg
     1501 ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg
     1561 ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc
     1621 tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc
     1681 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc
     1741 ctcgaggaat tcactcctca ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc
     1801 aatgccctgg ctcacaaata ccactgagat ctgcatgagc tagcgccacc atggtgagca
     1861 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1921 acggccacaa gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac ggcaagctga
     1981 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2041 ccttcggcta cggcgtggcc tgcttcagcc gctaccccga ccacatgaag cagcacgact
     2101 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctct ttcaaggacg
     2161 acggtaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2221 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2281 acaacttcaa cagccacaac gtctatatca cggccgacaa gcagaagaac ggcatcaagg
     2341 ctaacttcaa gatccgccac aacgttgagg acggcagcgt gcagctcgcc gaccactacc
     2401 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagcc
     2461 atcagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     2521 tcgtgaccgc cgccgggatt atgcaagttg ccgacgccag cgaagaggaa ctgagcgagt
     2581 gcttcagaat cttcgacttc gacggcaacg gcttcatcga cagagaggag tttggcgaca
     2641 tcatcagact gaccggcgag cagctgaccg acgaggacgt ggacgagatc ttcggcgact
     2701 ccgacaccga caagaacggc agaatcgatt tcgacgagtt cctgaagatg gtggaaaacg
     2761 tgcagcccat ctaccccgag ctcatggtct tcacactcga agatttcgtt ggggactggc
     2821 gacagacagc cggctacaac ctggaccaag tccttgaaca gggaggtgtg tccagtttgt
     2881 ttcagaatct cggggtgtcc gtaactccga tccaaaggat tgtcctgagc ggtgaaaatg
     2941 ggctgaagat cgacatccat gtcatcatcc cgtatgaagg tctgagcggc gaccaaatgg
     3001 gccagatcga aaaaattttt aaggtggtgt accctgtgga tgatcatcac tttaaggtga
     3061 tcctgcacta tggcacactg gtaatcgacg gggttacgcc gaacatgatc gactatttcg
     3121 gacggccgta tgaaggcatc gccgtgttcg acggcaaaaa gatcactgta acagggaccc
     3181 tttggaacgg caacaaaatt atcgacgagc gcctgatcaa ccccgacggc tccctgctgt
     3241 tccgagtaac catcaacgga gtgaccggct ggcggctgtg cgaacgcatt ctggcgtaag
     3301 gatccactag tccagtgtgg tggaattctg cagatatcca gcacagtggc ggccgctcga
     3361 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     3421 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3481 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3541 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3601 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3661 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3721 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3781 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3841 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3901 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3961 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     4021 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     4081 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     4141 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     4201 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4261 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4321 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     4381 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4441 aagctcccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc
     4501 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     4561 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     4621 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     4681 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     4741 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     4801 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     4861 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     4921 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     4981 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     5041 gtgcctgatt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     5101 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     5161 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     5221 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5281 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     5341 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5401 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5461 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5521 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5581 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5641 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5701 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5761 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5821 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5881 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5941 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6001 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6061 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6121 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6181 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6241 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6301 ccaccgctgg tagcggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6361 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6421 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6481 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6541 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6601 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6661 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6721 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6781 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6841 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6901 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6961 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7021 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7081 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7141 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7201 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7261 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7321 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7381 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7441 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7501 gcacatttcc ccgaaaagtg ccacctgacg tc
//
