LOCUS       sequence-395338-        6297 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Indirect detection of V5-tagged protein using the HaloTag protein 
            labeling technology..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6297)
  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 6297)
  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-HALOTAG
FEATURES             Location/Qualifiers
     source          1..6297
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        92..139
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             158..556
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    714..835
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(751..770)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     805..824
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(884..900)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(884..900)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(897..919)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    908..924
                     /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(932..938,939..956,957..962))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    977..998
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1115..1132)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1286..1874)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1366..1385)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2045..2836,2837..2905))
                     /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     2668..2687
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2906..3010)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3085..3104)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3276..3655
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3656..3859
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3810..3830
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3919..3928
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4300..5190
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             5209..5238
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5254..5271
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5294..5311)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5300..5524
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5570..5998
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5657..5676)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5867..5888
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6007..6027)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(6012..6297,1..44)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6192..6297,1..30)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6254..6273
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
       61 gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg catagtatat
      121 cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagcctt tgtctcaaga
      181 agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga
      241 ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt
      301 atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc
      361 ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag
      421 cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt
      481 gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta
      541 tgtgtgggag ggctaagcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt
      601 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
      661 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
      721 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
      781 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
      841 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
      901 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
      961 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     1021 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     1081 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     1141 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     1201 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     1261 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1321 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1381 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1441 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1501 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1561 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1621 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1681 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     1741 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1801 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1861 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1921 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     1981 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2041 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2101 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2161 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     2221 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     2281 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     2341 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2401 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2461 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     2521 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     2581 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     2641 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     2701 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     2761 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     2821 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     2881 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     2941 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3001 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     3061 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     3121 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     3181 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     3241 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
     3301 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     3361 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     3421 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     3481 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     3541 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     3601 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     3661 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     3721 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     3781 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     3841 ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggactca
     3901 gatctcgagc tcaagcttgc caccatggct gaagtgcagc tgcaagccag cggcggaggc
     3961 ttcgtgcagc ccggcggatc tctgagactg tcttgtgccg cttctggcac caccagcttc
     4021 ggcgatacaa tgggctggtt ccggcaggcc cctggaaagg aaagagagtt tgtgtccgcc
     4081 atctccagac agggcgacaa gcactactat gccgacagcg tgaaaggcag attcacaatc
     4141 agccgggaca acagcaagaa caccgtgtac ctgcagatga actctctgag agccgaagat
     4201 accgccacct actactgcgc cgagtggatg aataccagac gcgagttcat caccccttac
     4261 tggggccagg gaacacaggt gaccgtgtct agcggatcca tggcagaaat cggtactggc
     4321 tttccattcg acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt
     4381 ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg
     4441 tggcgcaaca tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc
     4501 ggtatgggca aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc
     4561 atggatgcct tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg
     4621 ggctccgctc tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca
     4681 tttatggagt tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag
     4741 accttccagg ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt
     4801 tttatcgagg gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac
     4861 cattaccgcg agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac
     4921 gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac
     4981 tggctgcacc agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc
     5041 ccaccggccg aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc
     5101 ggcccgggtc tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg
     5161 cgctggctgt cgacgctcga gatttccggc taatctagag ggcccttcga acaaaaactc
     5221 atctcagaag aggatctgaa tatgcatacc ggtcatcatc accatcacca ttgagtttaa
     5281 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     5341 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     5401 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     5461 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     5521 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     5581 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     5641 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     5701 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     5761 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     5821 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5881 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     5941 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     6001 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     6061 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     6121 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     6181 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     6241 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattcc
//
