LOCUS       sequence-395348-        6171 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BRET2 detection of ALFA-tagged protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6171)
  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 6171)
  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-GFP2
FEATURES             Location/Qualifiers
     source          1..6171
                     /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      908..917
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1013..1024)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             join(1307..1309,1310..1312,1313..2026)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNPHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLFTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1352..1373)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1613..1632)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1960..1981
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2042..2071
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2087..2104
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2127..2144)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2133..2357
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2403..2831
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2490..2509)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2700..2721
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2840..2860)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2845..3174
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3025..3160
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3087..3106
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3222..3269
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3288..3686
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3844..3965
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3881..3900)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3935..3954
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4014..4030)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4014..4030)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4027..4049)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4038..4054
                     /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(4062..4068,4069..4086,4087..4092))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4107..4128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4245..4262)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4416..5004)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4496..4515)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5175..5966,5967..6035))
                     /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     5798..5817
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6036..6140)
                     /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 gctggctagt
      901 taagcttgcc accatggcgt ccggggaagt tcaacttcaa gaaagcggtg gcggcttggt
      961 ccaaccgggt gggagcctcc gactttcatg tactgcttcc ggggtcacta tttctgccct
     1021 caatgcaatg gcgatgggct ggtataggca ggcacctgga gaaagaagag taatggttgc
     1081 ggcggtcagc gaaagaggaa acgctatgta ccgggaaagc gtgcagggac gctttaccgt
     1141 gaccagggat ttcaccaaca agatggtttc cttgcagatg gacaatttga aacccgaaga
     1201 cactgcggtg tattattgtc acgtcttgga agaccgagtg gattcatttc atgattattg
     1261 gggccagggt acccaagtca cggtgtcttc aggatccggt ggtgggatgg tgagcaaggg
     1321 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     1381 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     1441 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1501 gagctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1561 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     1621 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1681 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1741 ctacaacccc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     1801 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1861 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgttcaccca
     1921 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1981 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaatcta gagggccctt
     2041 cgaacaaaaa ctcatctcag aagaggatct gaatatgcat accggtcatc atcaccatca
     2101 ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2161 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2221 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2281 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2341 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2401 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2461 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2521 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2581 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2641 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2701 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2761 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2821 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2881 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     2941 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3001 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3061 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     3121 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     3181 ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg
     3241 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagcctt
     3301 tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca
     3361 tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg
     3421 gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg
     3481 ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg
     3541 gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca
     3601 aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc
     3661 cctctggtta tgtgtgggag ggctaagcac ttcgtggccg aggagcagga ctgacacgtg
     3721 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     3781 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     3841 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3901 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     3961 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     4021 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4081 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4141 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4201 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4261 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4321 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4381 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4441 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4501 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4561 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4621 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4681 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4741 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4801 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4861 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4921 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4981 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5041 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5101 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5161 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5221 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5281 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5341 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5401 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5461 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5521 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5581 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5641 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5701 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5761 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5821 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5881 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5941 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6001 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6061 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6121 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
