LOCUS       sequence-395224-        6162 bp    DNA     circular SYN 14-APR-2025
DEFINITION  BRET2 detection of V5-tagged protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6162)
  AUTHORS   Zeghal M, Matte K, Venes A, Patel S, Laroche G, Sarvan S, Joshi M, 
            Rain JC, Couture JF, Giguere PM
  TITLE     Development of a V5-tag-directed nanobody and its implementation as 
            an intracellular biosensor of GPCR signalling.
  JOURNAL   J Biol Chem. 2023 Jul 28:105107. doi: 10.1016/j.jbc.2023.105107.
  PUBMED    37517699
REFERENCE   2  (bases 1 to 6162)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol 
            Chem. 2023 Jul 28:105107. doi: 10.1016/j.jbc.2023.105107."
COMMENT     Sequence Label: NbV5-GFP2
FEATURES             Location/Qualifiers
     source          1..6162
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      908..917
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1298..1300,1301..1303,1304..2017)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNPHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLFTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1343..1364)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1604..1623)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1951..1972
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2033..2062
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2078..2095
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2118..2135)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2124..2348
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2394..2822
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2481..2500)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2691..2712
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2831..2851)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2836..3165
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3016..3151
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3078..3097
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3213..3260
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3279..3677
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3835..3956
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3872..3891)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3926..3945
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4005..4021)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4005..4021)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4018..4040)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4029..4045
                     /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(join(4053..4059,4060..4077,4078..4083))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4098..4119
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4236..4253)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4407..4995)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4487..4506)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5166..5957,5958..6026))
                     /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     5789..5808
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6027..6131)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagcttgcc accatggctg aagtgcagct gcaagccagc ggcggaggct tcgtgcagcc
      961 cggcggatct ctgagactgt cttgtgccgc ttctggcacc accagcttcg gcgatacaat
     1021 gggctggttc cggcaggccc ctggaaagga aagagagttt gtgtccgcca tctccagaca
     1081 gggcgacaag cactactatg ccgacagcgt gaaaggcaga ttcacaatca gccgggacaa
     1141 cagcaagaac accgtgtacc tgcagatgaa ctctctgaga gccgaagata ccgccaccta
     1201 ctactgcgcc gagtggatga ataccagacg cgagttcatc accccttact ggggccaggg
     1261 aacacaggtg accgtgtcta gcggatccgg tggtgggatg gtgagcaagg gcgaggagct
     1321 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1381 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1441 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgagctacgg
     1501 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1561 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1621 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1681 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaaccc
     1741 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     1801 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1861 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgttcaccc agtccgccct
     1921 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1981 cgggatcact ctcggcatgg acgagctgta caagtaatct agagggccct tcgaacaaaa
     2041 actcatctca gaagaggatc tgaatatgca taccggtcat catcaccatc accattgagt
     2101 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2161 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2221 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2281 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2341 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     2401 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2461 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2521 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2581 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2641 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2701 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2761 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2821 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     2881 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     2941 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3001 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3061 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3121 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     3181 tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata
     3241 tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagcct ttgtctcaag
     3301 aagaatccac cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag
     3361 actacagcgt cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg
     3421 tatatcattt tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg
     3481 cggcagctgg caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga
     3541 gcccctgcgg acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag
     3601 tgaaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt
     3661 atgtgtggga gggctaagca cttcgtggcc gaggagcagg actgacacgt gctacgagat
     3721 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     3781 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     3841 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3901 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     3961 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4021 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4081 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4141 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4201 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4261 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4321 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4381 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4441 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4501 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4561 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4621 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4681 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4741 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4801 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4861 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4921 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4981 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5041 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5101 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5161 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5221 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5281 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5341 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5401 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5461 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5521 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5581 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5641 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5701 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5761 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5821 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5881 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5941 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6001 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6061 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6121 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
