LOCUS       sequence-395382-        6392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the beta-arrestin1 containing a C-terminal ALFA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6392)
  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 6392)
  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: bArr1-ALFA
FEATURES             Location/Qualifiers
     source          1..6392
                     /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      922..931
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2180..2204)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2206..2244
                     /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"
     CDS             2263..2292
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2308..2325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2348..2365)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2354..2578
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2624..3052
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2711..2730)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2921..2942
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3061..3081)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3066..3395
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3246..3381
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3308..3327
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3443..3490
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3509..3907
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4065..4186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4102..4121)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4156..4175
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4235..4251)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4235..4251)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4248..4270)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4259..4275
                     /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(4283..4289,4290..4307,4308..4313))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4328..4349
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4466..4483)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4637..5225)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4717..4736)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5396..6187,6188..6256))
                     /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     6019..6038
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6257..6361)
                     /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 taagcttggc gcgccgctag cgccaccatg ggcgacaaag gcacgagagt gttcaagaag
      961 gcctctccta acggcaagct gaccgtgtac ctcggcaaga gggacttcgt ggaccacatc
     1021 gatctggtgg accctgtgga tggcgtggtg ctggtcgatc ccgagtacct gaaagaacgg
     1081 cgggtgtacg tgaccctgac ctgcgccttt agatacggcc gcgaggatct ggatgtgctg
     1141 ggcctgacct tcagaaagga cctgttcgtg gccaacgtgc agagcttccc accagctcca
     1201 gaggacaaga agcccctgac cagactgcaa gagcggctga ttaagaagct gggcgagcac
     1261 gcttacccct tcaccttcga gatccctcca aacctgcctt gcagcgtgac actccaacct
     1321 ggacctgagg ataccggaaa ggcctgtggc gtggactacg aagtgaaggc cttttgcgcc
     1381 gagaacctgg aagagaagat ccacaagcgg aacagcgtgc ggctggtcat cagaaaggtg
     1441 cagtatgccc ctgagaggcc cggacctcaa cctacagccg agacaaccag acagttcctg
     1501 atgagcgaca agcccctgca cctggaagcc agcctggaca aagagatcta ctaccacggc
     1561 gagcccatca gcgtgaacgt ccacgtgacc aacaacacca acaagaccgt gaagaaaatc
     1621 aagatctccg tgcggcagta cgccgacatc tgcctgttta acacagccca gtacaagtgc
     1681 cccgtggcca tggaagaggc cgatgataca gtggccccta gcagcacctt ctgcaaagtg
     1741 tataccctga cacctttcct ggccaacaac cgcgagaaga gaggactggc cctggacgga
     1801 aagctgaagc acgaggatac caacctggcc agcagcaccc tgcttagaga gggcgccaat
     1861 agagagatcc tgggcatcat cgtgtcctac aaagtgaaag tgaagctggt ggtgtccaga
     1921 ggcggcctgc ttggagatct ggcctcttct gatgtggccg tggaactgcc cttcacactg
     1981 atgcacccca agcctaaaga ggaacctcct cacagagagg tgcccgagaa cgagacaccc
     2041 gtggacacca atctgatcga gctggacaca aacgacgacg acatcgtgtt cgaggacttc
     2101 gcccggcaga gactgaaggg catgaaggac gacaaagagg aagaagagga cggcaccggc
     2161 tctccgcagc tcaataatcg gccaactttc ttgtacaaag tggttagtag actggaggag
     2221 gagctcagga ggcgacttac cgagtaatct agagggccct tcgaacaaaa actcatctca
     2281 gaagaggatc tgaatatgca taccggtcat catcaccatc accattgagt ttaaacccgc
     2341 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2401 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2461 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2521 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2581 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2641 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2701 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2761 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2821 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2881 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2941 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3001 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     3061 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3121 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3181 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3241 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3301 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3361 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3421 ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt
     3481 ataatacgac aaggtgagga actaaaccat ggccaagcct ttgtctcaag aagaatccac
     3541 cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt
     3601 cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt
     3661 tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg
     3721 caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg
     3781 acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag
     3841 tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga
     3901 gggctaagca cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca
     3961 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4021 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4081 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4141 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4201 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4261 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4321 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4381 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4441 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4501 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4561 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4621 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4681 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4741 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4801 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4861 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4921 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4981 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5041 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5101 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5161 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5221 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5281 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5341 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5401 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5461 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5521 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5581 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5641 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5701 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5761 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5821 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5881 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5941 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6001 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6061 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6121 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6181 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6241 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6301 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6361 gcacatttcc ccgaaaagtg ccacctgacg tc
//
