LOCUS       sequence-395342-        5977 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 5977)
  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 5977)
  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: NbAlfa-LgBit
FEATURES             Location/Qualifiers
     source          1..5977
                     /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(1022..1033)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1842..1883
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1893..1910
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1933..1950)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1939..2163
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2209..2637
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2296..2315)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2506..2527
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2646..2666)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2651..2980
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2831..2966
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2893..2912
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3028..3075
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3094..3492
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3650..3771
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3687..3706)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3741..3760
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3820..3836)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3820..3836)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3833..3855)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3844..3860
                     /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(3868..3874,3875..3892,3893..3898))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3913..3934
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4051..4068)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4222..4810)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4302..4321)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4981..5772,5773..5841))
                     /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     5604..5623
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5842..5946)
                     /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 ccatggcgtc cggggaagtt caacttcaag aaagcggtgg
      961 cggcttggtc caaccgggtg ggagcctccg actttcatgt actgcttccg gggtcactat
     1021 ttctgccctc aatgcaatgg cgatgggctg gtataggcag gcacctggag aaagaagagt
     1081 aatggttgcg gcggtcagcg aaagaggaaa cgctatgtac cgggaaagcg tgcagggacg
     1141 ctttaccgtg accagggatt tcaccaacaa gatggtttcc ttgcagatgg acaatttgaa
     1201 acccgaagac actgcggtgt attattgtca cgtcttggaa gaccgagtgg attcatttca
     1261 tgattattgg ggccagggta cccaagtcac ggtgtcttca ggatccggtg gtggcgggag
     1321 cggaggtgga gggtcgtcag gtgtcttcac actcgaagat ttcgttgggg actgggaaca
     1381 gacagccgcc tacaacctgg accaagtcct tgaacaggga ggtgtgtcca gtttgctgca
     1441 gaatctcgcc gtgtccgtaa ctccgatcca aaggattgtc cggagcggtg aaaatgccct
     1501 gaagatcgac atccatgtca tcatcccgta tgaaggtctg agcgccgacc aaatggccca
     1561 gatcgaagag gtgtttaagg tggtgtaccc tgtggatgat catcacttta aggtgatcct
     1621 gccctatggc acactggtaa tcgacggggt tacgccgaac atgctgaact atttcggacg
     1681 gccgtatgaa ggcatcgccg tgttcgacgg caaaaagatc actgtaacag ggaccctgtg
     1741 gaacggcaac aaaattatcg acgagcgcct gatcaccccc gacggctcca tgctgttccg
     1801 agtaaccatc aacagctaat ctagagggcc cgcggttcga aggtaagcct atccctaacc
     1861 ctctcctcgg tctcgattct acgcgtaccg gtcatcatca ccatcaccat tgagtttaaa
     1921 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     1981 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2041 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2101 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2161 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2221 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2281 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2341 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2401 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2461 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2521 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2581 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     2641 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2701 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2761 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     2821 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     2881 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     2941 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3001 atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc
     3061 atagtataat acgacaaggt gaggaactaa accatggcca agcctttgtc tcaagaagaa
     3121 tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc tgaagactac
     3181 agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt caatgtatat
     3241 cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc tgctgcggca
     3301 gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc
     3361 tgcggacggt gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc catagtgaag
     3421 gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg
     3481 tgggagggct aagcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga
     3541 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     3601 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     3661 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     3721 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     3781 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     3841 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     3901 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     3961 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4021 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4081 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4141 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4201 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4261 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4321 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4381 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4441 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4501 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4561 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4621 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     4681 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4741 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4801 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4861 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     4921 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     4981 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5041 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5101 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5161 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5221 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5281 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5341 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5401 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5461 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5521 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5581 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5641 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5701 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     5761 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     5821 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     5881 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     5941 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
