LOCUS       sequence-9771-ex        7471 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of opto-b2AR fused to EYFP for optical control 
            of GPCR signaling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7471)
  AUTHORS   Airan RD, Thompson KR, Fenno LE, Bernstein H, Deisseroth K
  TITLE     Temporally precise in vivo control of intracellular signalling.
  JOURNAL   Nature. 2009 Apr 23;458(7241):1025-9. doi: 10.1038/nature07926. Epub
            2009 Mar 18.
  PUBMED    19295515
REFERENCE   2  (bases 1 to 7471)
  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"; doi: 
            "10.1038/nature07926"; journalName: "Nature"; date: "2009-04-23- 
            23"; volume: "458"; issue: "7241"; pages: "1025-9"
COMMENT     Sequence Label: pcDNA3.1/opto-b2AR-EYFP
FEATURES             Location/Qualifiers
     source          1..7471
                     /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             929..967
                     /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             join(2222..2224,2225..2938)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2264..2285)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2525..2544)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2872..2893
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2968..3009
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3019..3036
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3059..3076)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3065..3289
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3335..3763
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3422..3441)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3632..3653
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3772..3792)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3777..4107
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3958..4093
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4020..4039
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4174..4968
                     /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(4228..4247)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4838..4857
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5144..5265
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5181..5200)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5235..5254
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5314..5330)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5314..5330)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5327..5349)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5338..5354
                     /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(5362..5368,5369..5386,5387..5392))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5407..5428
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5545..5562)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5716..6304)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5796..5815)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6475..7266,7267..7335))
                     /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     7098..7117
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7336..7440)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 taagcttgcc accatgaacg gaacagaggg cccaaacttt tacgttccct tctccaataa
      961 gactggggtc gtgagaagcc catttgaggc gcctcaatac taccttgctg agccgtggca
     1021 gttttctatg ctcgctgctt acatgttctt gctgatcatg ctggggttcc ctatcaattt
     1081 cctgacgctg tacgttatag caaagttcga acgcctccaa accgtgttga actacatact
     1141 ccttaacctc gcggttgccg acctcttcat ggttttcggg ggtttcacca ccaccctcta
     1201 cacctccctt cacggctact tcgtgttcgg ccctaccgga tgcaatctgg aaggcttttt
     1261 cgcaacgctg gggggggaga ttgccctttg gagcctggtg gtcttggcca tagagaggta
     1321 cgtggtggtc acatccccat tcaagtacca gagtttgctt acaaagaaca aggctatcat
     1381 gggggtcgcc ttcacatggg tgatggcgct ggcttgcgct gccccaccgc tggtaggctg
     1441 gtcccggtat attccggagg gaatgcagtg cagttgtggg atcgactact acaccccaca
     1501 cgaagagact aacaacgagt cttttgtgat ttatatgttc gtggtccact tcatcatccc
     1561 cctgatagtg atctttttct gttacggcag ggtgttccag gtcgccaaaa ggcagctcca
     1621 gaagatcgac aaaagcgaag gccgctttca cagccccaat cttggacagg ttgaacagga
     1681 cggcaggtca gggcacgggc tgcgacgcag ttctaagttc tgcctgaagg aacataaggc
     1741 cttgagaatg gtgatcatca tggtaatcgc cttcctgata tgctggcttc catacgctgg
     1801 cgtggctttt tatatattca cgcaccaggg gtcagatttt gggcctatct ttatgaccat
     1861 acctgctttc ttcgctaaga cgagtgcggt gtataaccca gtgatataca tcatgatgaa
     1921 caaacaattc agaattgcct tccaggaatt gctctgtctc agacgcagct cttccaaagc
     1981 gtacggaaat ggctattcat ctaacagcaa cggaaagact gattatatgg gcgaagccag
     2041 tggctgccag ctgggccagg aaaaagagag cgagcggctt tgtgaagatc ccccaggcac
     2101 tgagagcttc gtgaattgtc agggaacagt tccgagtctc tctcttgatt cacagggacg
     2161 caattgctct accaacgaca gccccctgga gacttcccag gtcgctccgg ccgcggccgc
     2221 cgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     2281 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     2341 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     2401 cgtgaccacc ttcggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca
     2461 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     2521 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     2581 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     2641 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     2701 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2761 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2821 cctgagctac cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2881 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaca
     2941 tatgctctag agggcccgcg gttcgaaggt aagcctatcc ctaaccctct cctcggtctc
     3001 gattctacgc gtaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc
     3061 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     3121 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     3181 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     3241 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     3301 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     3361 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3421 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3481 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3541 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3601 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3661 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttggggat ttcggcctat
     3721 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     3781 gtcagttagg gtgtggaaag tccccaggct ccccaggcag gcagaagtat gcaaagcatg
     3841 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3901 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3961 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     4021 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     4081 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     4141 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     4201 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     4261 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     4321 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     4381 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     4441 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     4501 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     4561 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     4621 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     4681 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     4741 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4801 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4861 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4921 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4981 ggttcgcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     5041 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     5101 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     5161 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     5221 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     5281 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5341 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5401 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5461 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5521 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5581 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5641 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5701 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5761 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5821 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5881 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt
     5941 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6001 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6061 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6121 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     6181 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6241 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6301 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6361 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6421 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6481 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6541 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6601 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6661 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6721 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6781 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6841 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6901 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6961 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     7021 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     7081 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     7141 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     7201 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7261 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7321 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7381 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7441 cacatttccc cgaaaagtgc cacctgacgt c
//
