LOCUS       sequence-395186-        6254 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 6254)
  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 6254)
  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: LgBit-NbV5
FEATURES             Location/Qualifiers
     source          1..6254
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        253..456
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     407..427
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     501..520
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        501..519
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      555..564
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1483..1500)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1489..1713
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1759..2187
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1846..1865)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2056..2077
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2196..2216)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2201..2530
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2381..2516
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2443..2462
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2597..3391
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2651..2670)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3261..3280
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3565..3686
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3602..3621)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3656..3675
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3735..3751)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3735..3751)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3748..3770)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3759..3775
                     /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(3783..3789,3790..3807,3808..3813))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3828..3849
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3966..3983)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4137..4725)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4217..4236)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4896..5687,5688..5756))
                     /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     5519..5538
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5757..5861)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5936..5955)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6127..6254,1..252)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
       61 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      121 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      181 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      241 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      301 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      361 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      421 aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact
      481 gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc tggctagcgt
      541 ttaaacttaa gcttgccacc atggtcttca cactcgaaga tttcgttggg gactgggaac
      601 agacagccgc ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc agtttgctgc
      661 agaatctcgc cgtgtccgta actccgatcc aaaggattgt ccggagcggt gaaaatgccc
      721 tgaagatcga catccatgtc atcatcccgt atgaaggtct gagcgccgac caaatggccc
      781 agatcgaaga ggtgtttaag gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc
      841 tgccctatgg cacactggta atcgacgggg ttacgccgaa catgctgaac tatttcggac
      901 ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt
      961 ggaacggcaa caaaattatc gacgagcgcc tgatcacccc cgacggctcc atgctgttcc
     1021 gagtaaccat caacagtggg agttccggtg gtggcgggag cggaggtgga ggctcgagca
     1081 tggctgaagt gcagctgcaa gccagcggcg gaggcttcgt gcagcccggc ggatctctga
     1141 gactgtcttg tgccgcttct ggcaccacca gcttcggcga tacaatgggc tggttccggc
     1201 aggcccctgg aaaggaaaga gagtttgtgt ccgccatctc cagacagggc gacaagcact
     1261 actatgccga cagcgtgaaa ggcagattca caatcagccg ggacaacagc aagaacaccg
     1321 tgtacctgca gatgaactct ctgagagccg aagataccgc cacctactac tgcgccgagt
     1381 ggatgaatac cagacgcgag ttcatcaccc cttactgggg ccagggaaca caggtgaccg
     1441 tgtctagcta atctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     1501 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     1561 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     1621 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     1681 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     1741 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     1801 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     1861 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     1921 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     1981 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2041 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2101 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2161 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     2221 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2281 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2341 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     2401 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2461 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2521 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     2581 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     2641 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     2701 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     2761 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     2821 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     2881 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     2941 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     3001 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     3061 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     3121 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     3181 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     3241 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     3301 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     3361 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     3421 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3481 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3541 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     3601 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3661 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     3721 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3781 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3841 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3901 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3961 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4021 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4081 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4141 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4201 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4261 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4321 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4381 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4441 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4501 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4561 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4621 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4681 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4741 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4801 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4861 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4921 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4981 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5041 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5101 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5161 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5221 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5281 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5341 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5401 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5461 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5521 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5581 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5641 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5701 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5761 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5821 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5881 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     5941 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     6001 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     6061 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
     6121 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     6181 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     6241 cgcccaacga cccc
//
