LOCUS       sequence-395347-        6288 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NanoBit detection of ALFA-tagged protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6288)
  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 6288)
  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-NbALFA
FEATURES             Location/Qualifiers
     source          1..6288
                     /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             complement(1541..1552)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1879..1896)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1885..2109
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2155..2583
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2242..2261)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2452..2473
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2592..2612)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2597..2926
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2777..2912
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2839..2858
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2993..3787
                     /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(3047..3066)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3657..3676
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3961..4082
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3998..4017)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4052..4071
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4131..4147)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4131..4147)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4144..4166)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4155..4171
                     /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(4179..4185,4186..4203,4204..4209))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4224..4245
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4362..4379)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4533..5121)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4613..4632)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5292..6083,6084..6152))
                     /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     5915..5934
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6153..6257)
                     /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 ccatggtctt cacactcgaa gatttcgttg gggactggga
      961 acagacagcc gcctacaacc tggaccaagt ccttgaacag ggaggtgtgt ccagtttgct
     1021 gcagaatctc gccgtgtccg taactccgat ccaaaggatt gtccggagcg gtgaaaatgc
     1081 cctgaagatc gacatccatg tcatcatccc gtatgaaggt ctgagcgccg accaaatggc
     1141 ccagatcgaa gaggtgttta aggtggtgta ccctgtggat gatcatcact ttaaggtgat
     1201 cctgccctat ggcacactgg taatcgacgg ggttacgccg aacatgctga actatttcgg
     1261 acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa cagggaccct
     1321 gtggaacggc aacaaaatta tcgacgagcg cctgatcacc cccgacggct ccatgctgtt
     1381 ccgagtaacc atcaacagtg ggagttccgg tggtggcggg agcggaggtg gaggctcgag
     1441 catggcgtcc ggggaagttc aacttcaaga aagcggtggc ggcttggtcc aaccgggtgg
     1501 gagcctccga ctttcatgta ctgcttccgg ggtcactatt tctgccctca atgcaatggc
     1561 gatgggctgg tataggcagg cacctggaga aagaagagta atggttgcgg cggtcagcga
     1621 aagaggaaac gctatgtacc gggaaagcgt gcagggacgc tttaccgtga ccagggattt
     1681 caccaacaag atggtttcct tgcagatgga caatttgaaa cccgaagaca ctgcggtgta
     1741 ttattgtcac gtcttggaag accgagtgga ttcatttcat gattattggg gccagggtac
     1801 ccaagtcacg gtgtcttcat aagaattcag tctaagctag cagatcttct agagggcccg
     1861 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     1921 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     1981 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     2041 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     2101 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     2161 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2221 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2281 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2341 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2401 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2461 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2521 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2581 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     2641 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     2701 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     2761 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     2821 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     2881 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     2941 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     3001 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3061 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3121 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     3181 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     3241 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     3301 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     3361 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     3421 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3481 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     3541 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     3601 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     3661 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     3721 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     3781 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     3841 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     3901 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     3961 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4021 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4081 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     4141 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4201 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4261 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4321 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4381 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4441 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4501 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4561 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4621 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4681 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4741 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4801 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4861 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4921 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4981 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5041 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5101 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5161 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5221 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5281 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5341 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5401 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5461 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5521 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5581 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5641 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5701 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5761 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5821 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5881 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5941 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6001 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6061 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6121 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6181 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6241 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
