LOCUS       sequence-395156-        5968 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NanoBit detection of V5-tagged protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5968)
  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 5968)
  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"; journalName: "J Biol 
            Chem. 2023 Jul 28:105107. doi: 10.1016/j.jbc.2023.105107."
COMMENT     Sequence Label: NbV5-LgBit
FEATURES             Location/Qualifiers
     source          1..5968
                     /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      917..926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1833..1874
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1884..1901
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1924..1941)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1930..2154
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2200..2628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2287..2306)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2497..2518
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2637..2657)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2642..2971
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2822..2957
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2884..2903
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3019..3066
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3085..3483
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3641..3762
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3678..3697)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3732..3751
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3811..3827)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3811..3827)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3824..3846)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3835..3851
                     /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(3859..3865,3866..3883,3884..3889))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3904..3925
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4042..4059)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4213..4801)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4293..4312)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4972..5763,5764..5832))
                     /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     5595..5614
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5833..5937)
                     /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 gctggctagc
      901 gtttaaactt aagcttgcca ccatggctga agtgcagctg caagccagcg gcggaggctt
      961 cgtgcagccc ggcggatctc tgagactgtc ttgtgccgct tctggcacca ccagcttcgg
     1021 cgatacaatg ggctggttcc ggcaggcccc tggaaaggaa agagagtttg tgtccgccat
     1081 ctccagacag ggcgacaagc actactatgc cgacagcgtg aaaggcagat tcacaatcag
     1141 ccgggacaac agcaagaaca ccgtgtacct gcagatgaac tctctgagag ccgaagatac
     1201 cgccacctac tactgcgccg agtggatgaa taccagacgc gagttcatca ccccttactg
     1261 gggccaggga acacaggtga ccgtgtctag cggatccggt ggtggcggga gcggaggtgg
     1321 agggtcgtca ggtgtcttca cactcgaaga tttcgttggg gactgggaac agacagccgc
     1381 ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc agtttgctgc agaatctcgc
     1441 cgtgtccgta actccgatcc aaaggattgt ccggagcggt gaaaatgccc tgaagatcga
     1501 catccatgtc atcatcccgt atgaaggtct gagcgccgac caaatggccc agatcgaaga
     1561 ggtgtttaag gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgccctatgg
     1621 cacactggta atcgacgggg ttacgccgaa catgctgaac tatttcggac ggccgtatga
     1681 aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa
     1741 caaaattatc gacgagcgcc tgatcacccc cgacggctcc atgctgttcc gagtaaccat
     1801 caacagctaa tctagagggc ccgcggttcg aaggtaagcc tatccctaac cctctcctcg
     1861 gtctcgattc tacgcgtacc ggtcatcatc accatcacca ttgagtttaa acccgctgat
     1921 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     1981 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2041 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     2101 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     2161 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     2221 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2281 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2341 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2401 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2461 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2521 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     2581 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     2641 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2701 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     2761 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     2821 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     2881 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     2941 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     3001 cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     3061 tacgacaagg tgaggaacta aaccatggcc aagcctttgt ctcaagaaga atccaccctc
     3121 attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc
     3181 agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact
     3241 gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac
     3301 ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg
     3361 tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat
     3421 ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc
     3481 taagcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc
     3541 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     3601 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     3661 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3721 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     3781 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3841 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3901 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3961 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4021 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4081 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4141 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4201 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4261 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4321 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4381 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4441 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4501 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4561 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4621 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4681 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4741 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4801 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4861 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4921 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4981 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5041 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5101 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5161 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5221 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5281 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5341 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5401 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5461 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5521 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5581 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5641 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5701 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5761 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5821 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5881 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5941 atttccccga aaagtgccac ctgacgtc
//
