LOCUS       sequence-395411-        6635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the beta-arrestin2 containing a N-terminal ALFA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6635)
  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 6635)
  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: ALFA-bArr2
FEATURES             Location/Qualifiers
     source          1..6635
                     /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"
     CDS             926..964
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     primer_bind     complement(2226..2243)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2232..2456
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2502..2930
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2589..2608)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2799..2820
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2939..2959)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2944..3273
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3124..3259
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3186..3205
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3340..4134
                     /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(3394..3413)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4004..4023
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4308..4429
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4345..4364)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4399..4418
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4478..4494)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4478..4494)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4491..4513)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4502..4518
                     /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(4526..4532,4533..4550,4551..4556))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4571..4592
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4709..4726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4880..5468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4960..4979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5639..6430,6431..6499))
                     /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     6262..6281
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6500..6604)
                     /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 ccatgagtag actggaggag gagctcagga ggcgacttac
      961 cgagatgggg gagaaacccg ggaccagggt cttcaagaag tcgagcccta actgcaagct
     1021 caccgtgtac ttgggcaagc gggacttcgt agatcacctg gacaaagtgg accctgtaga
     1081 tggcgtggtg cttgtggacc ctgactacct gaaggaccgc aaagtgtttg tgaccctcac
     1141 ctgcgccttc cgctatggcc gtgaagacct ggatgtgctg ggcttgtcct tccgcaaaga
     1201 cctgttcatc gccacctacc aggccttccc cccggtgccc aacccacccc ggccccccac
     1261 ccgcctgcag gaccggctgc tgaggaagct gggccagcat gcccacccct tcttcttcac
     1321 cataccccag aatcttccat gctccgtcac actgcagcca ggcccagagg atacaggaaa
     1381 ggcctgcggc gtagactttg agattcgagc cttctgtgct aaatcactag aagagaaaag
     1441 ccacaaaagg aactctgtgc ggctggtgat ccgaaaggtg cagttcgccc cggagaaacc
     1501 cggcccccag ccttcagccg aaaccacacg ccacttcctc atgtctgacc ggtccctgca
     1561 cctcgaggct tccctggaca aggagctgta ctaccatggg gagcccctca atgtaaatgt
     1621 ccacgtcacc aacaactcca ccaagaccgt caagaagatc aaagtctctg tgagacagta
     1681 cgccgacatc tgcctcttca gcaccgccca gtacaagtgt cctgtggctc aactcgaaca
     1741 agatgaccag gtatctccca gctccacatt ctgtaaggtg tacaccataa ccccactgct
     1801 cagtgacaac cgggagaagc ggggtctcgc cctggatggg aaactcaagc acgaggacac
     1861 caacctggct tccagcacca tcgtgaagga gggtgccaac aaggaggtgc tgggaatcct
     1921 ggtgtcctac agggtcaagg tgaagctggt ggtgtctcga ggcggggatg tctctgtgga
     1981 gctgcctttt gttcttatgc accccaagcc ccacgaccac atccccctcc ccagacccca
     2041 gtcagccgct ccggagacag atgtccctgt ggacaccaac ctcattgaat ttgataccaa
     2101 ctatgccaca gatgatgaca ttgtgtttga ggactttgcc cggcttcggc tgaaggggat
     2161 gaaggatgac gactatgatg atcaactctg ctaatctaga gggcccgttt aaacccgctg
     2221 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2281 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2341 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2401 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2461 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     2521 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2581 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2641 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2701 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     2761 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2821 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2881 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     2941 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3001 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3061 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3121 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3181 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     3241 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     3301 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3361 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3421 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3481 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     3541 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3601 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3661 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3721 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3781 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3841 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     3901 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3961 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4021 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4081 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4141 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4201 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4261 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4321 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4381 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4441 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4501 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4561 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4621 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4681 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4741 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4801 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4861 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4921 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4981 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5041 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5101 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5161 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5221 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5281 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5341 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5401 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5461 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5521 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5581 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5641 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5701 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5761 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5821 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5881 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5941 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6001 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6061 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6121 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6181 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6241 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6301 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6361 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6421 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6481 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6541 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6601 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
