LOCUS       sequence-237157-        8138 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of cannabinoid receptor CB2 fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8138)
  AUTHORS   Calandra Bernard, Julie Tucker, David Shire, Reinhard Grisshammer
  TITLE     Expression in Escherichia coli and characterisation of the human 
            central CB1 and peripheral CB2 cannabinoid receptors
  JOURNAL   Biotechnology Letters
REFERENCE   2  (bases 1 to 8138)
  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: 
            "Biotechnology Letters"
COMMENT     Sequence Label: pRG/III-hs-MBP-CB2-HF
FEATURES             Location/Qualifiers
     source          1..8138
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     44..66
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(115..192,193..1290)
                     /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      1147..1156
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1264..1287
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2383..2400
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2407..2430
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2476..2493)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2476..2492)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2485..2507)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2671..2688)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2841..2927
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3019..3046
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3065..3156
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3157..3225,3226..4017)
                     /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(3375..3394)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     5085..5104
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5302..5890
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5791..5810
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6044..6061
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6227..6249
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6243..6434)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6930..7007
                     /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             7008..8090
                     /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(7027..7046)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    8103..8124
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgacc atgattacga atttctagat aacgagggca aaccatgaaa
      121 ataaaaacag gtgcacgcat cctcgcatta tccgcattaa cgacgatgat gttttccgcc
      181 tcggctctcg ccaaaatcga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc
      241 tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc
      301 gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc
      361 cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg
      421 gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc
      481 gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt
      541 tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat
      601 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc
      661 acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac
      721 gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt
      781 gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc
      841 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac
      901 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa
      961 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca
     1021 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac
     1081 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag tgttggcgaa agatccacgt
     1141 attgccgcca ccatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg
     1201 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact
     1261 gtcgatgaag ccctgaaaga cgcgcagact ggatccgagg aatgctgggt gacagagata
     1321 gccaatggct ccaaggatgg cttggattcc aaccctatga aggattacat gatcctgagt
     1381 ggtccccaga agacagctgt tgctgtgttg tgcactcttc tgggcctgct aagtgccctg
     1441 gagaacgtgg ctgtgctcta tctgatcctg tcctcccacc aactccgccg gaagccctca
     1501 tacctgttca ttggcagctt ggctggggct gacttcctgg ccagtgtggt ctttgcatgc
     1561 agctttgtga atttccatgt tttccatggt gtggattcca aggctgtctt cctgctgaag
     1621 attggcagcg tgactatgac cttcacagcc tctgtgggta gcctcctgct gaccgccatt
     1681 gaccgatacc tctgcctgcg ctatccacct tcctacaaag ctctgctcac ccgtggaagg
     1741 gcactggtga ccctgggcat catgtgggtc ctctcagcac tagtctccta cctgcccctc
     1801 atgggatgga cttgctgtcc caggccctgc tctgagcttt tcccactgat ccccaatgac
     1861 tacctgctga gctggctcct gttcatcgcc ttcctctttt ccggaatcat ctacacctat
     1921 gggcatgttc tctggaaggc ccatcagcat gtggccagct tgtctggcca ccaggacagg
     1981 caggtgccag gaatggcccg aatgaggctg gatgtgaggt tggccaagac cctagggcta
     2041 gtgttggctg tgctcctcat ctgttggttc ccagtgctgg ccctcatggc ccacagcctg
     2101 gccactacgc tcagtgacca ggtcaagaag gcctttgctt tctgctccat gctgtgcctc
     2161 atcaactcca tggtcaaccc tgtcatctat gctctacgga gtggagagat ccgctcctct
     2221 gcccatcact gcctggctca ctggaagaag tgtgtgaggg gccttgggtc agaggcaaaa
     2281 gaagaagccc cgagatcctc agtcaccgag acagaggctg atgggaaaat cactccgtgg
     2341 ccagattcca gagatctaga cctctctgat tgcgcggccg cacaccatca ccatcaccat
     2401 ggtaccgact acaaggacga cgatgacaag gaattctaat aatctagaac cggttaataa
     2461 ctcgagaagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     2521 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     2581 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcagcttgg
     2641 ctgttttggc ggatgagata agattttcag cctgatacag attaaatcag aacgcagaag
     2701 cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat
     2761 gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag
     2821 agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     2881 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg
     2941 atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg
     3001 ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa
     3061 ctcttttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3121 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3181 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3241 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3301 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgtttccc aatgatgagc
     3361 acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa
     3421 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3481 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3541 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3601 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3661 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     3721 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3781 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3841 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     3901 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     3961 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4021 tcagaccaag tttactcata tatactttag attgatttac cccggttgat aatcagaaaa
     4081 gccccaaaaa caggaagatt gtataagcaa atatttaaat tgtaaacgtt aatattttgt
     4141 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     4201 gcaaaatccc ttataaatca aaagaatagc ccgagatccg gccgaacaaa ctccgggagg
     4261 cagcgtgatg cggcaacaat cacacggatt tcccgtgaac ggtctgaatg agcggattat
     4321 tttcagggaa agtgagtgtg gtcagcgtgc aggtatatgg gctatgatgt gcccggcgct
     4381 tgaggctttc tgcctcatga cgtgaaggtg gtttgttgcc gtgttgtgtg gcagaaagaa
     4441 gatagccccg tagtaagtta attttcatta accaccacga ggcatcccta tgtctagtcc
     4501 acatcaggat agcctcttac cgcgctttgc gcaaggagaa gaaggccatg aaactaccac
     4561 gaagttccct tgtctggtgt gtgttgatcg tgtgtctcac actgttgata ttcacttatc
     4621 tgacacgaaa atcgctgtgc gagattcgtt acagagacgg acacagggag gtggcggctt
     4681 tcatggctta cgaatccggt aagtagcaac ctggaggcgg gcgcaggccc gccttttcag
     4741 gactgatgct ggtctgacta ctgaagcgcc tttataaagg ggctgctggt tcgccggtag
     4801 cccctttctc cttgctgatg ttgtgggaat tcctgaatcg ccccatcatc cagccagaaa
     4861 gtgagggagc cacggttgat gagagctttg ttgtaggtgg accagttggt gattttgaac
     4921 ttttgctttg ccacggaacg gtctgcgttg tcgggaagat gtgaaccatc acccaaatca
     4981 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     5041 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     5101 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     5161 gccgcgctta atgcgccgct acagggcgcg taaaaggatc taggtgaaga tcctttttga
     5221 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5281 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5341 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5401 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     5461 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5521 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5581 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5641 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5701 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5761 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5821 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5881 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5941 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     6001 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     6061 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     6121 ccgcataggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     6181 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     6241 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     6301 gcgtggtcgt gcagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     6361 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     6421 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     6481 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     6541 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     6601 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     6661 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     6721 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     6781 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     6841 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     6901 ggccaggacc caacgctgcc cgaaattccg acaccatcga atggtgcaaa acctttcgcg
     6961 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
     7021 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     7081 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     7141 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     7201 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     7261 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     7321 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     7381 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     7441 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     7501 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     7561 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     7621 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     7681 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     7741 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     7801 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
     7861 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     7921 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     7981 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     8041 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     8101 attaatgtga gttagctcac tcattaggca ccccaggc
//
