LOCUS       sequence-328558-       10264 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Membrane expression detection of Cannabinoid receptor 1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10264)
  AUTHORS   Rodriguez-Rodriguez I, Kalafut J, Czerwonka A, Rivero-Muller A
  TITLE     A novel bioassay for quantification of surface Cannabinoid receptor 
            1 expression.
  JOURNAL   Sci Rep. 2020 Oct 23;10(1):18191. doi: 10.1038/s41598-020-75331-y.
  PUBMED    33097803
REFERENCE   2  (bases 1 to 10264)
  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: "Sci Rep."; 
            date: "2020-10-23"; volume: "10(1):18191. doi"; pages: " 
            10.1038/s41598-020-75331-y"
COMMENT     Sequence Label: GLuc-FLAG-CB1
FEATURES             Location/Qualifiers
     source          1..10264
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1003
                     /label=chicken beta-actin promoter
     intron          1004..1231
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1372..1923
                     /codon_start=1
                     /product="secreted Gaussia luciferase"
                     /label=hGLuc
                     /note="human codon-optimized"
                     /translation="GVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRGK
                     LPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQ
                     GGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQR
                     CATFASKIQGQVDKIKGAGGD"
     CDS             1933..1956
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     repeat_region   7527..7667
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7527..7656
                     /label=AAV2 ITR
     rep_origin      7742..8197
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7829..7848)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8039..8060
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8214..8233)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8333..8355
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8393..8411)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8479..8583
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8584..8652,8653..9444)
                     /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(8802..8821)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9615..10203
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10104..10123
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc cctccccacc
      781 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
      841 gggggggcgc gcgccrgggg gggggggggg gggggggggg gggggggggg sgaggcggag
      901 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg
      961 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg
     1021 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac
     1081 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct
     1141 gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct
     1201 ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgcgaccat gatcaaaata
     1261 gccacacgta aatatttagg caaacaaaat gtctatgaca ttggagttga gcgcgaccat
     1321 aattttgcac tcaaaaatgg cttcatagct tctaattgtt tcaatggtac cggagtcaaa
     1381 gttctgtttg ccctgatctg catcgctgtg gccgaggcca agcccaccga gaacaacgaa
     1441 gacttcaaca tcgtggccgt ggccagcaac ttcgcgacca cggatctcga tgctgaccgc
     1501 gggaagttgc ccggcaagaa gctgccgctg gaggtgctca aagagatgga agccaatgcc
     1561 cggaaagctg gctgcaccag gggctgtctg atctgcctgt cccacatcaa gtgcacgccc
     1621 aagatgaaga agttcatccc aggacgctgc cacacctacg aaggcgacaa agagtccgca
     1681 cagggcggca taggcgaggc gatcgtcgac attcctgaga ttcctgggtt caaggacttg
     1741 gagcccatgg agcagttcat cgcacaggtc gatctgtgtg tggactgcac aactggctgc
     1801 ctcaaagggc ttgccaacgt gcagtgttct gacctgctca agaagtggct gccgcaacgc
     1861 tgtgcgacct ttgccagcaa gatccagggc caggtggaca agatcaaggg ggccggtggt
     1921 gacggactcg aggattacaa ggatgacgac gataagaagt cgatcctaga tggccttgca
     1981 gataccacct tccgcaccat caccactgac ctcctgtacg tgggctcaaa tgacattcag
     2041 tacgaagaca tcaaaggtga catggcatcc aaattagggt acttcccaca gaaattccct
     2101 ttaacttcct ttaggggaag tcccttccaa gagaagatga ctgcgggaga caacccccag
     2161 ctagtcccag cagaccaggt gaacattaca gaattttaca acaagtctct ctcgtccttc
     2221 aaggagaatg aggagaacat ccagtgtggg gagaacttca tggacataga gtgtttcatg
     2281 gtcctgaacc ccagccagca gctggccatt gcagtcctgt ccctcacgct gggcaccttc
     2341 acggtcctgg agaacctcct ggtgctgtgc gtcatcctcc actcccgcag cctccgctgc
     2401 aggccttcct accacttcat cggcagcctg gcggtggcag acctcctggg gagtgtcatt
     2461 tttgtctaca gcttcattga cttccacgtg ttccaccgca aagatagccg caacgtgttt
     2521 ctgttcaaac tgggtggggt cacggcctcc ttcactgcct ccgtgggcag cctgttcctc
     2581 acagccatcg acaggtacat atccattcac aggcccctgg cctataagag gattgtcacc
     2641 aggcccaagg ccgtggtggc gttttgcctg atgtggacca tagccattgt gatcgccgtg
     2701 ctgcctctcc tgggctggaa ctgcgagaaa ctgcaatctg tttgctcaga cattttccca
     2761 cacattgatg aaacctacct gatgttctgg atcggggtca ccagcgtact gcttctgttc
     2821 atcgtgtatg cgtacatgta tattctctgg aaggctcaca gccacgccgt ccgcatgatt
     2881 cagcgtggca cccagaagag catcatcatc cacacgtctg aggatgggaa ggtacaggtg
     2941 acccggccag accaagcccg catggacatt aggttagcca agaccctggt cctgatcctg
     3001 gtggtgttga tcatctgctg gggccctctg cttgcaatca tggtgtatga tgtctttggg
     3061 aagatgaaca agctcattaa gacggtgttt gcattctgca gtatgctctg cctgctgaac
     3121 tccaccgtga accccatcat ctatgctctg aggagtaagg acctgcgaca cgctttccgg
     3181 agcatgtttc cctcttgtga aggcactgcg cagcctctgg ataacagcat gggggactcg
     3241 gactgcctgc acaaacacgc aaacaatgca gccagtgttc acagggccgc agaaagctgc
     3301 atcaagagca cggtcaagat tgccaaggta accatgtctg tgtccacaga cacgtctgcc
     3361 gaggctctgt gagcctgatg cctccctggc agcacaggaa aagaattttt tttttaagct
     3421 caaaatctag aagagtctat tgtctccttg gttatatttt tttaacttta ccatgctcaa
     3481 tgaaaaggtg attgtcacca tgatcactta tcagtttgct aatgtttcca tagtttaggt
     3541 actcaaactc cattctccag gggtttacag tgaagaaagc ctgttgttta agtgactgaa
     3601 cgatccttca aagtctcaat gaaataggag ggaaaccttt ggctacacaa ttggaagtct
     3661 aagaacccat ggaaaaatgc catcaaatga ataatgcctt tgtaaccaca actttcacta
     3721 taatgtgaaa tgtaactgtc cgtagtatca gagatgtcca tttttacaag ttatagtact
     3781 agagatattt tgtaaaatgt attatgtcct gtgagatgtg tatcagtgtt tatgtgctat
     3841 taatatttgt ttagttcagc aaaactgaaa ggtagacttt tatgagaaca atggacaagc
     3901 agtggatacg tgtcaatgtg tgcacttttt ttctatatta ttgcccatga tataacttta
     3961 gaaataaacc ttaatatttc ttcaaatatc tctatttaat tttgacactg aaataaccgt
     4021 aaaggtttat ttttctgtta cctcaacaag aagaatttga agacttcaaa atattgagca
     4081 gaattcattc atacttaaaa atttattagc cctgcatttt cataggaaga cacattatct
     4141 tctggactat agctgttcta atggattata atcagaatgg aagagagaaa gcatattgac
     4201 tttttttgag cgacatctct gactttcttt agtctttagc tattactgga tctcttaaga
     4261 cagcatgtgt taatcttaat gtatatcgtt atcactgtgc agttgctgtt tacttgaata
     4321 gtattgtgtt cctatattcc aggtttaagt agatttcatg cctgggtggc caaacaacag
     4381 tcttcatttt ttttaattga aaagaagtag tgtctggatc agtaaaatta tactgtgtgt
     4441 gagtgtgaat ataaatgtgt gtatgtgtgt ttctgtcctg taactgttac agtaatgtca
     4501 taaagtgaga aaactgtgac caagtataaa cttttaccac ttgctgcact cttgcacatg
     4561 gattcagttt ctaaaattga gttcttcctg taatcttgtt gataaaaata ctgactccaa
     4621 ccattcaaaa atttcacccc atccctcctt aagagattgg atcaagtatt actaaattga
     4681 cctttaggta ttacacaaga ccagtgctta gcaaaaaata atgacaggca tccaaggaag
     4741 ggatgtattt gtagtgttat tgccaggaaa ggagagtact ttggtttctg agcaccgaat
     4801 attgagcaat atgtcagtca ctaaaaggaa gacagttcta cagaaaaaca atggtaacat
     4861 ttttcaatag cgtgtgtaga tagtatgcac tatatacatc acgttaaagt aggactatca
     4921 cacccagccc atgtggctaa aaaagctgaa tcagacagtg gatgagacac acaacggcag
     4981 tgaagaaccg atacacttgg cattgacgtc tagctatgct gtatctgtgc tttgcccaca
     5041 tgcccttggt gacagctgag cacccagctc tgtcttggta ggtttgggct aaggaacaaa
     5101 tctctccttt gctcgtggtt agcaagatac actcaagcat gaagataaac acagctgctt
     5161 tcttcttaca ccccggtctc atgctcctta atggcgccat gggtgcttgt tgggcctttt
     5221 tccagtaagg aatgatattg ctgaagaatc tacttaaccc tgacaaattt taattataat
     5281 ctcttcttat acagataaaa catgactcct acaaggcccc aaggtttaca tagtctgaag
     5341 tgaagtacag agctggcatc tatctggtga tttctagctc tcgagatacc caagcagcct
     5401 gatggggcag ttccccttct tacggttcac gctctaaggc aggatgtggc ttatgagata
     5461 ctttgcattg tctgtctgca caccttgaat ctgcctgctg gctcccttac tttacctctc
     5521 tgtcatgtgc agatgaaggc tcagggtgct agaggattag taagatctct ttctaaagac
     5581 aggagagatt atttacaaga agaactcacc agggtttagt ttgcatttaa gaattgccag
     5641 tcttttgtcc tgcatcatct tgaacattaa tccacatgtt tcagagctca ccaggcagta
     5701 ccaatgctct tttcacagct atgaagagct agagaaattc ttgttatggt agaaaaattt
     5761 cacgattcat ttttgaaact gcatttgtgc gtatgcagtg tagattttat agtgtgttgt
     5821 gctttcaaga tctaaatcat atataataaa ttaagggaca atggggctga cagcactaaa
     5881 cttggtgctt attgatattc taagaaatat ctgtgaaata tcatcacgta tgttatacaa
     5941 ccttcattta aaaaggttta aaactagtta gattcacttt gacacttttc atatcatttc
     6001 ttaacccaag tgacgaaaac attgtcccca atgaatatac tcattagaat taccatttgt
     6061 taatatcact cattaattaa ccccataatt agatccatta atttaaatga tttaaattta
     6121 agtaagtttt ataaggtctg acatcagagg tatcttactt tcctctgagg atgatgtact
     6181 tgccctgacc atgcatttta ccatcacaca tgttcagaaa gggccaaatt cccaacctgc
     6241 tcattttttt ttttatcaga gtcatgatga atcagtccta gaatgtttca tttgcacaag
     6301 tagggctgcc tccaagagga acctctgatt tattttgtat gaaatatatg tgaaaggata
     6361 tgaatctgag agatgctgta gacatctgtc ctacacttga gatgatttcc aagcctctct
     6421 ggcactttga gttaagtcta tctggtatta aatgccaagg accttttgct gcctaaatcc
     6481 actctgcagg aaataggccc aaccaccaga tgagaattag gccctggatg agtagcgcta
     6541 tagttactgt cctgttgatt aatttctgcc atttcatgtc cataaaagag accacccata
     6601 tcatgcacac aattagattt ctcacactct aactgtatat ttgtatgata ttttaaaatc
     6661 tcctaaatgc tgggcaatgg ctattaacaa ttaattgtct tgcactggcc ttctgatgaa
     6721 atgttaacaa tgcctattgt aatatagaaa aaaacattct atctactgat ttgggctgaa
     6781 tgtatgtaaa taggtttcta aaaagtcaga tgtttgagca gtggcctaca aatcagtaat
     6841 tttcggatgg gagagtttct ttacattgcc gtggcatctt aaaagctatc ttcatgtaaa
     6901 ttgactgtac taggcctact ggggatcaga gttcccaaga aaggaaacct tttcttgtat
     6961 ctggattcaa atttatttcc aatgtttcaa gcgggaaaca tgactcttta ttgtctgtaa
     7021 atctaacatt attacttttc ctcttagaag aatattgtat tgttagatgt ttgttgagct
     7081 ggtaacatcg ttgcaaccac tgcaatatct tcgttagtaa tctgtataat actttgtata
     7141 caagtactgg taagattgtt attaaatgta gcttcagtca ttaaattact atagcaaagt
     7201 agtacttctt ctgtaatatt tacaatgtat taagcccaca gtatatttta tttcaatgta
     7261 attaaactgt taacttattc aaagagaaaa catctcatca tgtctattgt ccaaagttac
     7321 ctggaatcaa ataaaaattc tagattacca tgaagaacat aaaatgcctt tgaactctgc
     7381 cttatttcac agtctgatgg caaaaaatga ggaaattgca tcgcattgtc tgagtaggtg
     7441 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa
     7501 tagcaggcat gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc
     7561 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     7621 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     7681 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     7741 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     7801 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     7861 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     7921 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     7981 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     8041 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     8101 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     8161 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     8221 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     8281 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     8341 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     8401 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     8461 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     8521 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     8581 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     8641 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     8701 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     8761 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     8821 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     8881 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     8941 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     9001 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     9061 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     9121 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     9181 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     9241 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga
     9301 agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     9361 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     9421 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     9481 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     9541 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     9601 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     9661 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     9721 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     9781 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     9841 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     9901 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     9961 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
    10021 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
    10081 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
    10141 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
    10201 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
    10261 atgt
//
