LOCUS       sequence-373322-        8026 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of ADRA2A with mEGFP tag on N-terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8026)
  AUTHORS   Foust DJ, Piston DW
  TITLE     Measuring G Protein Activation with Spectrally Resolved Fluorescence
            Fluctuation Spectroscopy
  JOURNAL   bioRxiv 2021.11.03.467169
REFERENCE   2  (bases 1 to 8026)
  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: "bioRxiv 
            2021.11.03.467169"
COMMENT     Sequence Label: pmEGFP-ADRA2A
FEATURES             Location/Qualifiers
     source          1..8026
                     /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      1021..1030
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1027..1029,1030..1032,1033..1740)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(1072..1093)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1333..1352)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1680..1701
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3448..3465)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3454..3678
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3724..4152
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3811..3830)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4021..4042
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4161..4181)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4166..4495
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4346..4481
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4408..4427
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4544..5569
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4544..5569
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    5699..5820
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5736..5755)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5790..5809
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5869..5885)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5869..5885)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5882..5904)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5893..5909
                     /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(5917..5923,5924..5941,5942..5947))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5962..5983
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6100..6117)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6271..6859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6351..6370)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7030..7821,7822..7890))
                     /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     7653..7672
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7891..7995)
                     /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 gctggctaga
      901 ctcgagggcc gcccagcttc caccatgggc ctccgggcgc tgatgctgtg gttgctggcg
      961 gcgacggggc tcgtgcgcga gtccctgcaa ggagagttcc aaaggaagct gtacccggtc
     1021 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1081 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     1141 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1201 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     1261 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     1321 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1381 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1441 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1501 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1561 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1621 aaccactacc tgagcaccca gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac
     1681 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1741 atgttccggc aggagcagcc actcgccgag ggttcctttg cccccatggg aagtcttcag
     1801 cctgacgccg gcaacgctag ctggaatggc accgaagcgc ccggtggagg ggctcgggct
     1861 accccatatt ctctccaggt cacactgacc ctggtttgtc tggccggtct cctgatgctg
     1921 ctgacggtct ttggcaatgt tttggtgata attgcggtgt tcacctccag agcactcaag
     1981 gctccccaga acctgttctt ggtcagcctg gcgtctgccg atattctggt cgccaccctc
     2041 gtaattccgt tttctcttgc caatgaggtt atgggttact ggtattttgg taaggcatgg
     2101 tgcgagatat accttgctct cgacgttctc ttctgcacca gcagtattgt gcatctgtgc
     2161 gcaattagtc tggacagata ttggtccatc acgcaggcaa tagaatataa cttgaaaagg
     2221 actcctcggc gcattaaggc tatcatcatc actgtctggg tcattagcgc tgtgataagc
     2281 ttccctccac ttatttccat tgaaaaaaaa ggaggaggcg gagggccgca gcctgcggag
     2341 ccccgctgtg agatcaacga tcagaaatgg tatgtcatct cctcctgcat tgggtccttc
     2401 ttcgccccat gtctgatcat gatcctcgta tatgttagga tttatcagat agctaagcga
     2461 aggacccggg tccccccatc caggagaggt ccagacgcag tggccgctcc ccccggaggc
     2521 acagaaagga gacctaacgg tctgggacca gagcgctccg caggacctgg aggtgcagaa
     2581 gccgagccct tgccgacaca actgaacgga gctcccggcg aaccagcccc agcaggtcct
     2641 cgcgatactg acgcactgga cctcgaagag tcctctagca gcgatcatgc agagcgccct
     2701 ccagggccta ggagacctga aagaggaccc agaggtaaag gcaaggcccg ggcaagtcaa
     2761 gtgaaaccgg gagactctct gcctcggagg ggtccaggtg ctactggtat agggactcct
     2821 gccgccggtc ccggagaaga gcgggtcggc gccgctaagg caagtcgctg gcggggcaga
     2881 caaaaccggg aaaaaaggtt tacattcgtg ctcgcggtag tgattggcgt cttcgtcgtg
     2941 tgctggtttc cattcttttt tacctatacc ctgacagccg tcggctgttc tgttccacgg
     3001 acactgttca agttcttttt ctggtttggc tactgtaact caagcctgaa cccggtaatc
     3061 tatacaatat ttaaccacga ttttcggagg gcttttaaaa agatcctgtg cagaggcgat
     3121 aggaaacgaa tcgtttgact ctgctgcctg cccgcacagc agcctgcttc ccacctccct
     3181 gcccaggcca gccagcctca cccttgcgaa ccgtgagcag gaaggcctgg gtggatcggc
     3241 ctcctcttca ccccggcagg ccctgcagtg ttcgcttggc tccatgctcc tcactgcccg
     3301 cacaccctca ctctgccagg gcagtgctag tgagctgggc atggtaccga gctcggatcc
     3361 actagtccag tgtggtggaa ttctgcagat atccagcaca gtggcggccg ctcgagtcta
     3421 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     3481 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     3541 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3601 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3661 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     3721 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3781 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3841 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3901 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3961 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4021 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     4081 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     4141 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     4201 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     4261 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4321 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4381 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     4441 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     4501 cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgatgaaaa agcctgaact
     4561 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat
     4621 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     4681 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca
     4741 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
     4801 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     4861 cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga
     4921 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     4981 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     5041 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     5101 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     5161 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     5221 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     5281 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     5341 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     5401 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     5461 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     5521 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtgctacg
     5581 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     5641 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     5701 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     5761 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5821 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5881 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5941 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     6001 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6061 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6121 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6181 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6241 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6301 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6361 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6421 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6481 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6541 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6601 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6661 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6721 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6781 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6841 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6901 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6961 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     7021 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7081 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7141 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7201 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7261 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7321 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7381 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7441 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7501 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7561 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7621 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7681 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7741 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7801 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7861 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7921 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7981 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
