LOCUS       sequence-236050-        7956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of adenosine A2a receptor fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7956)
  AUTHORS   Weiss HM, Grisshammer R
  TITLE     Purification and characterization of the human adenosine A(2a) 
            receptor functionally expressed in Escherichia coli.
  JOURNAL   Eur J Biochem. 2002 Jan;269(1):82-92.
  PUBMED    11784301
REFERENCE   2  (bases 1 to 7956)
  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: "Eur J 
            Biochem."; date: "2002-01"; volume: "269(1)"; pages: "82-92"
COMMENT     Sequence Label: pRG/III-hs-MBP-A2aTr316-H10
FEATURES             Location/Qualifiers
     source          1..7956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(36..113,114..1211)
                     /codon_start=1
                     /gene="malE"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This gene encodes a signal sequence that can direct 
                     MBP to the bacterial periplasm."
                     /translation="MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNG
                     LAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAE
                     ITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDK
                     ELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLV
                     DLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPS
                     KPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEVLAKD
                     PRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1068..1077
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1185..1208
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2172..2201
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(2215..2232)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2215..2231)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2224..2246)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2410..2427)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2580..2666
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2758..2785
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2804..2895
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2896..2964,2965..3756)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3114..3133)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     4824..4843
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5041..5629
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5530..5549
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5783..5800
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5966..5988
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5982..6173)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6669..6746
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             6747..7829
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(6766..6785)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    7842..7863
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7878..7883,7884..7901,7902..7908)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7916..7932
                     /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)."
     primer_bind     7921..7943
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7940..7956
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7940..7956
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 catgattacg aatttctaga taacgagggc aaaccatgaa aataaaaaca ggtgcacgca
       61 tcctcgcatt atccgcatta acgacgatga tgttttccgc ctcggctctc gccaaaatcg
      121 aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt ctcgctgaag
      181 tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat ccggataaac
      241 tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt atcttctggg
      301 cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc accccggaca
      361 aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac aacggcaagc
      421 tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa gatctgctgc
      481 cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg aaagcgaaag
      541 gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg ctgattgctg
      601 ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa gacgtgggcg
      661 tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt aaaaacaaac
      721 acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa ggcgaaacag
      781 cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa gtgaattatg
      841 gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga
      901 gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc ctcgaaaact
      961 atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag
     1021 cgctgaagtc ttacgaggaa gtgttggcga aagatccacg tattgccgcc accatggaaa
     1081 acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc tggtatgccg
     1141 tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag
     1201 acgcgcagac tggatccccc atcatgggct cctcggtgta catcacggtg gagctggcca
     1261 ttgctgtgct ggccatcctg ggcaatgtgc tggtgtgctg ggccgtgtgg ctcaacagca
     1321 acctgcagaa cgtcaccaac tactttgtgg tgtcactggc ggcggccgac atcgcagtgg
     1381 gtgtgctcgc catccccttt gccatcacca tcagcaccgg gttctgcgct gcctgccacg
     1441 gctgcctctt cattgcctgc ttcgtcctgg tcctcacgca gagctccatc ttcagtctcc
     1501 tggccatcgc cattgaccgc tacattgcca tccgcatccc gctccggtac aatggcttgg
     1561 tgaccggcac gagggctaag ggcatcattg ccatctgctg ggtgctgtcg tttgccatcg
     1621 gcctgactcc catgctaggt tggaacaact gcggtcagcc aaaggagggc aagaaccact
     1681 cccagggctg cggggagggc caagtggcct gtctctttga ggatgtggtc cccatgaact
     1741 acatggtgta cttcaacttc tttgcctgtg tgctggtgcc cctgctgctc atgctgggtg
     1801 tctatttgcg gatcttcctg gcggcgcgac gacagctgaa gcagatggag agccagcctc
     1861 tgccggggga gcgggcacgg tccacactgc agaaggaggt ccatgctgcc aagtcactgg
     1921 ccatcattgt ggggctcttt gccctctgct ggctgcccct acacatcatc aactgcttca
     1981 ctttcttctg ccccgactgc agccacgccc ctctctggct catgtacctg gccatcgtcc
     2041 tctcccacac caattcggtt gtgaatccct tcatctacgc ctaccgtatc cgcgagttcc
     2101 gccagacctt ccgcaagatc attcgcagcc acgtcctgag gcagcaagaa cctttcaagg
     2161 cagcggccgc acaccatcac catcaccatc accatcacca ttaataagct tggcactggc
     2221 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     2281 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     2341 ccaacagttg cgcagcctga atggcgaatg gcagcttggc tgttttggcg gatgagataa
     2401 gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt
     2461 gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg
     2521 ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc
     2581 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     2641 tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac
     2701 ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga
     2761 aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttttgttt atttttctaa
     2821 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2881 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2941 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3001 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3061 gagagttttc gccccgaaga acgtttccca atgatgagca cttttaaagt tctgctatgt
     3121 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     3181 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3241 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3301 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3361 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3421 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3481 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3541 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3601 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3661 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3721 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3781 atactttaga ttgatttacc ccggttgata atcagaaaag ccccaaaaac aggaagattg
     3841 tataagcaaa tatttaaatt gtaaacgtta atattttgtt aaaattcgcg ttaaattttt
     3901 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     3961 aagaatagcc cgagatccgg ccgaacaaac tccgggaggc agcgtgatgc ggcaacaatc
     4021 acacggattt cccgtgaacg gtctgaatga gcggattatt ttcagggaaa gtgagtgtgg
     4081 tcagcgtgca ggtatatggg ctatgatgtg cccggcgctt gaggctttct gcctcatgac
     4141 gtgaaggtgg tttgttgccg tgttgtgtgg cagaaagaag atagccccgt agtaagttaa
     4201 ttttcattaa ccaccacgag gcatccctat gtctagtcca catcaggata gcctcttacc
     4261 gcgctttgcg caaggagaag aaggccatga aactaccacg aagttccctt gtctggtgtg
     4321 tgttgatcgt gtgtctcaca ctgttgatat tcacttatct gacacgaaaa tcgctgtgcg
     4381 agattcgtta cagagacgga cacagggagg tggcggcttt catggcttac gaatccggta
     4441 agtagcaacc tggaggcggg cgcaggcccg ccttttcagg actgatgctg gtctgactac
     4501 tgaagcgcct ttataaaggg gctgctggtt cgccggtagc ccctttctcc ttgctgatgt
     4561 tgtgggaatt cctgaatcgc cccatcatcc agccagaaag tgagggagcc acggttgatg
     4621 agagctttgt tgtaggtgga ccagttggtg attttgaact tttgctttgc cacggaacgg
     4681 tctgcgttgt cgggaagatg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc
     4741 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     4801 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     4861 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     4921 cagggcgcgt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4981 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5041 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5101 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5161 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     5221 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5281 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5341 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5401 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5461 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5521 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5581 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5641 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5701 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     5761 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     5821 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatagggt catggctgcg
     5881 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     5941 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     6001 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg cagcgattca
     6061 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     6121 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     6181 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     6241 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     6301 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     6361 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     6421 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     6481 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     6541 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     6601 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gccaggaccc aacgctgccc
     6661 gaaattccga caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     6721 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
     6781 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     6841 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     6901 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     6961 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     7021 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     7081 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     7141 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     7201 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
     7261 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     7321 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
     7381 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
     7441 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     7501 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg
     7561 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     7621 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     7681 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     7741 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     7801 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     7861 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     7921 agcggataac aatttcacac aggaaacagc tatgac
//
