LOCUS       sequence-244586-        6662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SPARK o-beta-2AR transmembrane component in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6662)
  AUTHORS   Kim CK, Cho KF, Kim MW, Ting AY
  TITLE     Luciferase-LOV BRET enables versatile and specific transcriptional 
            readout of cellular protein-protein interactions.
  JOURNAL   eLife. 2019 Apr 3;8. pii: 43826. doi: 10.7554/eLife.43826.
  PUBMED    30942168
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: "eLife."; 
            date: "2019-04-3"; volume: "8. pii: 43826. doi"; pages: " 
            10.7554/eLife.43826"
COMMENT     Sequence Label: pAAV-gLuc sp-GS
            linker-HiBit-Flag-o-beta-2AR-TS-NNE...
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        201..504
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        505..708
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     659..679
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     753..772
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        753..771
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      785..794
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             905..928
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             941..979
                     /codon_start=1
                     /product="segment of bovine rhodopsin containing a signal 
                     for N-linked glycosylation (Favaloro et al., 2008)"
                     /label=opsin tag
                     /note="The asparagine in the NKT tripeptide will be 
                     glycosylated if this sequence is exposed to the ER lumen."
                     /translation="GPNFYVPFSNKTG"
     CDS             2768..2788
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     CDS             2801..2824
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2825..3262
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="KLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKR
                     SPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKD
                     AVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             3263..3604
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="activation domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 activation domain
                     /translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     CDS             3605..3646
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3683..3700)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3689..3896
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3925..4065
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3925..4054
                     /label=AAV2 ITR
     rep_origin      4140..4595
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4227..4246)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4437..4458
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4612..4631)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4731..4753
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4791..4809)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4877..4981
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4982..5050,5051..5842)
                     /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     complement(5200..5219)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6013..6601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6502..6521
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgttgtaca tagtaatcaa ttacggggtc attagttcat
      181 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      241 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      301 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      361 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      421 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      481 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      541 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      601 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      661 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      721 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      781 gcttgccacc atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc
      841 cggctcgggc agcggtggct ctggaggttc tgtctcaggg tggaggctct ttaagaagat
      901 cagtgactac aaggatgacg acgataagaa cggaacagag ggcccaaact tttacgttcc
      961 cttctccaat aagactgggg tcgtgagaag cccatttgag gcgcctcaat actaccttgc
     1021 tgagccgtgg cagttttcta tgctcgctgc ttacatgttc ttgctgatca tgctggggtt
     1081 ccctatcaat ttcctgacgc tgtacgttat agcaaagttc gaacgcctcc aaaccgtgtt
     1141 gaactacata ctccttaacc tcgcggttgc cgacctcttc atggttttcg ggggtttcac
     1201 caccaccctc tacacctccc ttcacggcta cttcgtgttc ggccctaccg gatgcaatct
     1261 ggaaggcttt ttcgcaacgc tgggggggga gattgccctt tggagcctgg tggtcttggc
     1321 catagagagg tacgtggtgg tcacatcccc attcaagtac cagagtttgc ttacaaagaa
     1381 caaggctatc atgggggtcg ccttcacatg ggtgatggcg ctggcttgcg ctgccccacc
     1441 gctggtaggc tggtcccggt atattccgga gggaatgcag tgcagttgtg ggatcgacta
     1501 ctacacccca cacgaagaga ctaacaacga gtcttttgtg atttatatgt tcgtggtcca
     1561 cttcatcatc cccctgatag tgatcttttt ctgttacggc agggtgttcc aggtcgccaa
     1621 aaggcagctc cagaagatcg acaaaagcga aggccgcttt cacagcccca atcttggaca
     1681 ggttgaacag gacggcaggt cagggcacgg gctgcgacgc agttctaagt tctgcctgaa
     1741 ggaacataag gccttgagaa tggtgatcat catggtaatc gccttcctga tatgctggct
     1801 tccatacgct ggcgtggctt tttatatatt cacgcaccag gggtcagatt ttgggcctat
     1861 ctttatgacc atacctgctt tcttcgctaa gacgagtgcg gtgtataacc cagtgatata
     1921 catcatgatg aacaaacaat tcagaattgc cttccaggaa ttgctctgtc tcagacgcag
     1981 ctcttccaaa gcgtacggaa atggctattc atctaacagc aacggaaaga ctgattatat
     2041 gggcgaagcc agtggctgcc agctgggcca ggaaaaagag agcgagcggc tttgtgaaga
     2101 tcccccaggc actgagagct tcgtgaattg tcagggaaca gttccgagtc tctctcttga
     2161 ttcacaggga cgcaattgct ctaccaacga cagccccctg gagacttccc aggtcgctcc
     2221 ggccaagagc aggatcacca gcgagggcga gtacatcccc ctggaccaga tcgacatcaa
     2281 cgtgactagt gagctggccg agaagctggc cggcctggac atcaacggtg gagctagcgg
     2341 cagccgggcg accaccctgg aacgtattga aaagagcttt gtgattaccg atccgcgtct
     2401 gccggataac ccgattattt ttgtgagcga tagctttctg cagctgaccg aatatagccg
     2461 tgaagaaatt ctgggccgta actgccgttt tctgcagggc ccggaaaccg atcgtgcgac
     2521 cgtgcgtaaa attcgtgatg cgattgataa ccagaccgaa gtgaccgtgc agctgattaa
     2581 ctataccaaa agcggcaaaa aattttggaa cctgtttcat ctgcagccga tgcgtgatca
     2641 gaaaggcgat gtgcagtatt ttattggcgt gcagctggat ggtaccgaac gtgtgcgtga
     2701 tgcggcggaa cgtgaagctg tgatgctggt caagaaaacc gcggaggaaa tcgacgaagc
     2761 cgctaaggaa aatctctact tccagatggg cggaggatcc gactacaagg atgacgacga
     2821 taagaagcta ctgtcttcta tcgaacaagc atgcgatatt tgccgactta aaaagctcaa
     2881 gtgctccaaa gaaaaaccga agtgcgccaa gtgtctgaag aacaactggg agtgtcgcta
     2941 ctctcccaaa accaaaaggt ctccgctgac tagggcacat ctgacagaag tggaatcaag
     3001 gctagaaaga ctggaacagc tatttctact gatttttcct cgagaagacc ttgacatgat
     3061 tttgaaaatg gattctttac aggatataaa agcattgtta acaggattat ttgtacaaga
     3121 taatgtgaat aaagatgccg tcacagatag attggcttca gtggagactg atatgcctct
     3181 aacattgaga cagcatagaa taagtgcgac atcatcatcg gaagagagta gtaacaaagg
     3241 tcaaagacag ttgactgtat cggccaattt taatcaaagt gggaatattg ctgatagctc
     3301 attgtccttc actttcacta acagtagcaa cggtccgaac ctcataacaa ctcaaacaaa
     3361 ttctcaagcg ctttcacaac caattgcctc ctctaacgtt catgataact tcatgaataa
     3421 tgaaatcacg gctagtaaaa ttgatgatgg taataattca aaaccactgt cacctggttg
     3481 gacggaccaa actgcgtata acgcgtttgg aatcactaca gggatgttta ataccactac
     3541 aatggatgat gtatataact atctattcga tgatgaagat accccaccaa acccaaaaaa
     3601 agagggcaag ccgatcccta accctctgct gggcctggac agcacttagt aagaattcct
     3661 cgagctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     3721 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     3781 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     3841 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggaggta
     3901 accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     3961 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     4021 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     4081 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     4141 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4201 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4261 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4321 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     4381 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4441 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     4501 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4561 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     4621 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     4681 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4741 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     4801 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     4861 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4921 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4981 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5041 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5101 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5161 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5221 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5281 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5341 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5401 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5461 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5521 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5581 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5641 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag
     5701 ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5761 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5821 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5881 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5941 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6001 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6061 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6121 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6181 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6241 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6301 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6361 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6421 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6481 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6541 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6601 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6661 gt
//
