LOCUS       sequence-395415-        6656 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 6656)
  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 6656)
  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: bArr2-ALFA
FEATURES             Location/Qualifiers
     source          1..6656
                     /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"
     protein_bind    complement(2148..2172)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2174..2212
                     /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(2247..2264)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2253..2477
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2523..2951
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2610..2629)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2820..2841
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2960..2980)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2965..3294
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3145..3280
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3207..3226
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3361..4155
                     /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(3415..3434)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4025..4044
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4329..4450
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4366..4385)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4420..4439
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4499..4515)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4499..4515)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4512..4534)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4523..4539
                     /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(4547..4553,4554..4571,4572..4577))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4592..4613
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4730..4747)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4901..5489)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4981..5000)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5660..6451,6452..6520))
                     /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     6283..6302
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6521..6625)
                     /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 ccatggggga gaaacccggg accagggtct tcaagaagtc
      961 gagccctaac tgcaagctca ccgtgtactt gggcaagcgg gacttcgtag atcacctgga
     1021 caaagtggac cctgtagatg gcgtggtgct tgtggaccct gactacctga aggaccgcaa
     1081 agtgtttgtg accctcacct gcgccttccg ctatggccgt gaagacctgg atgtgctggg
     1141 cttgtccttc cgcaaagacc tgttcatcgc cacctaccag gccttccccc cggtgcccaa
     1201 cccaccccgg ccccccaccc gcctgcagga ccggctgctg aggaagctgg gccagcatgc
     1261 ccaccccttc ttcttcacca taccccagaa tcttccatgc tccgtcacac tgcagccagg
     1321 cccagaggat acaggaaagg cctgcggcgt agactttgag attcgagcct tctgtgctaa
     1381 atcactagaa gagaaaagcc acaaaaggaa ctctgtgcgg ctggtgatcc gaaaggtgca
     1441 gttcgccccg gagaaacccg gcccccagcc ttcagccgaa accacacgcc acttcctcat
     1501 gtctgaccgg tccctgcacc tcgaggcttc cctggacaag gagctgtact accatgggga
     1561 gcccctcaat gtaaatgtcc acgtcaccaa caactccacc aagaccgtca agaagatcaa
     1621 agtctctgtg agacagtacg ccgacatctg cctcttcagc accgcccagt acaagtgtcc
     1681 tgtggctcaa ctcgaacaag atgaccaggt atctcccagc tccacattct gtaaggtgta
     1741 caccataacc ccactgctca gtgacaaccg ggagaagcgg ggtctcgccc tggatgggaa
     1801 actcaagcac gaggacacca acctggcttc cagcaccatc gtgaaggagg gtgccaacaa
     1861 ggaggtgctg ggaatcctgg tgtcctacag ggtcaaggtg aagctggtgg tgtctcgagg
     1921 cggggatgtc tctgtggagc tgccttttgt tcttatgcac cccaagcccc acgaccacat
     1981 ccccctcccc agaccccagt cagccgctcc ggagacagat gtccctgtgg acaccaacct
     2041 cattgaattt gataccaact atgccacaga tgatgacatt gtgtttgagg actttgcccg
     2101 gcttcggctg aaggggatga aggatgacga ctatgatgat caactctgcc caactttctt
     2161 gtacaaagtg gttagtagac tggaggagga gctcaggagg cgacttaccg agtaatctag
     2221 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2281 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2341 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2401 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2461 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2521 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2581 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2641 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2701 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2761 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2821 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2881 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2941 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3001 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3061 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3121 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3181 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     3241 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     3301 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     3361 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3421 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3481 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3541 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3601 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3661 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3721 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3781 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3841 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     3901 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3961 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4021 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4081 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4141 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4201 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4261 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4321 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4381 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4441 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4501 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4561 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4621 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4681 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4741 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4801 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4861 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4921 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4981 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5041 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5101 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5161 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5221 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5281 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5341 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5401 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5461 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5521 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5581 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5641 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5701 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5761 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5821 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5881 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5941 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6001 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6061 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6121 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6181 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6241 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6301 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6361 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6421 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6481 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6541 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6601 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
