LOCUS       sequence-395380-        6662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the beta-arrestin1 containing a N-terminal ALFA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6662)
  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 6662)
  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-bArr1
FEATURES             Location/Qualifiers
     source          1..6662
                     /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(2253..2270)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2259..2483
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2529..2957
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2616..2635)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2826..2847
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2966..2986)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2971..3300
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3151..3286
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3213..3232
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3367..4161
                     /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(3421..3440)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4031..4050
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4335..4456
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4372..4391)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4426..4445
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4505..4521)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4505..4521)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4518..4540)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4529..4545
                     /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(4553..4559,4560..4577,4578..4583))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4598..4619
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4736..4753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4907..5495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4987..5006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5666..6457,6458..6526))
                     /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     6289..6308
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6527..6631)
                     /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 cgagatgggc gacaaaggca cgagagtgtt caagaaggcc tctcctaacg gcaagctgac
     1021 cgtgtacctc ggcaagaggg acttcgtgga ccacatcgat ctggtggacc ctgtggatgg
     1081 cgtggtgctg gtcgatcccg agtacctgaa agaacggcgg gtgtacgtga ccctgacctg
     1141 cgcctttaga tacggccgcg aggatctgga tgtgctgggc ctgaccttca gaaaggacct
     1201 gttcgtggcc aacgtgcaga gcttcccacc agctccagag gacaagaagc ccctgaccag
     1261 actgcaagag cggctgatta agaagctggg cgagcacgct taccccttca ccttcgagat
     1321 ccctccaaac ctgccttgca gcgtgacact ccaacctgga cctgaggata ccggaaaggc
     1381 ctgtggcgtg gactacgaag tgaaggcctt ttgcgccgag aacctggaag agaagatcca
     1441 caagcggaac agcgtgcggc tggtcatcag aaaggtgcag tatgcccctg agaggcccgg
     1501 acctcaacct acagccgaga caaccagaca gttcctgatg agcgacaagc ccctgcacct
     1561 ggaagccagc ctggacaaag agatctacta ccacggcgag cccatcagcg tgaacgtcca
     1621 cgtgaccaac aacaccaaca agaccgtgaa gaaaatcaag atctccgtgc ggcagtacgc
     1681 cgacatctgc ctgtttaaca cagcccagta caagtgcccc gtggccatgg aagaggccga
     1741 tgatacagtg gcccctagca gcaccttctg caaagtgtat accctgacac ctttcctggc
     1801 caacaaccgc gagaagagag gactggccct ggacggaaag ctgaagcacg aggataccaa
     1861 cctggccagc agcaccctgc ttagagaggg cgccaataga gagatcctgg gcatcatcgt
     1921 gtcctacaaa gtgaaagtga agctggtggt gtccagaggc ggcctgcttg gagatctggc
     1981 ctcttctgat gtggccgtgg aactgccctt cacactgatg caccccaagc ctaaagagga
     2041 acctcctcac agagaggtgc ccgagaacga gacacccgtg gacaccaatc tgatcgagct
     2101 ggacacaaac gacgacgaca tcgtgttcga ggacttcgcc cggcagagac tgaagggcat
     2161 gaaggacgac aaagaggaag aagaggacgg caccggctct ccgcagctca ataatcggta
     2221 atctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     2281 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2341 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2401 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2461 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     2521 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2581 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2641 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2701 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2761 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2821 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2881 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2941 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3001 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3061 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3121 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3181 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     3241 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3301 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     3361 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     3421 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     3481 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     3541 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     3601 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     3661 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     3721 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     3781 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     3841 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     3901 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     3961 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4021 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4081 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4141 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     4201 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4261 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4321 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4381 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4441 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     4501 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4561 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4621 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4681 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4741 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4801 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4861 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4921 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4981 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5041 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5101 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5161 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5221 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5281 gagcgaggta tgtaggcggt gctacagagt tcttgaaatg gtggcctaac tacggctaca
     5341 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5401 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5461 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5521 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5581 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5641 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5701 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5761 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5821 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5881 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5941 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6001 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6061 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6121 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6181 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6241 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6301 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6361 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6421 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6481 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6541 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6601 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6661 tc
//
