LOCUS       sequence-177101-        7482 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses CB1R-iTango component in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7482)
  AUTHORS   Lee D, Creed M, Jung K, Stefanelli T, Wendler DJ, Oh WC, Mignocchi 
            NL, Luscher C, Kwon HB
  TITLE     Temporally precise labeling and control of neuromodulatory circuits 
            in the mammalian brain.
  JOURNAL   Nat Methods. 2017 May;14(5):495-503. doi: 10.1038/nmeth.4234. Epub 
            2017 Apr 3.
  PUBMED    28369042
REFERENCE   2  (bases 1 to 7482)
  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/nmeth.4234"; journalName: "Nat Methods"; date: "2017-05"; 
            volume: "14"; issue: "5"; pages: "495-503"
COMMENT     Sequence Label: pCMV-CB1R-V2tail-TevN-BLITz1-TetR-VP16
FEATURES             Location/Qualifiers
     source          1..7482
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2438..2458
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2459..3076,3077..3463)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTD
                     SMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIE
                     GLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHAD
                     ALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(2529..2548)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3472..3491)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3473..3491)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3496..3630
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3520..3539)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3574..3593
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3743..3763)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3743..3761)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3782..3798)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3782..3798)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3795..3817)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3806..3822
                     /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(3830..3836,3837..3854,3855..3860))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3875..3896
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4013..4030)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4183..4771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4263..4282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4942..5733,5734..5802))
                     /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     5565..5584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5803..5907)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5933..6388)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6070..6091)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6282..6301
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6459..7443
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 atgaagacga tcatcgccct gagctacatc ttctgcctgg tattcgccga ctacaaggac
       61 gatgatgacg ccagcatcga tatgaagagc attttggatg ggctcgctga taccacattc
      121 cggacaatta caactgatct gctgtacgtg ggttcaaacg atattcagta tgaggacata
      181 aaaggcgata tggcctctaa actgggctat ttcccacaga agttccctct caccagcttc
      241 cgcggtagcc cttttcagga gaaaatgact gcgggagaca atcctcagct cgtccccgca
      301 gaccaggtga acattactga attttacaac aagagtttga gcagctttaa ggagaacgag
      361 gagaacatac agtgcggaga aaactttatg gacattgagt gctttatggt gctgaatccc
      421 agtcagcagc tggctatagc ggtgctgtcc ctgacccttg ggactttcac ggtcttggag
      481 aatctgctgg tgctgtgcgt catcttgcac agccggtcac tgcggtgtcg gccaagttat
      541 cactttattg gctcactcgc tgtcgctgat ctcctgggct ccgtgatttt cgtttattca
      601 ttcatcgact tccatgtctt ccaccggaaa gactcacgga acgtttttct tttcaagctt
      661 ggaggcgtta cggccagttt tactgctagc gtaggaagcc tgttcctgac tgcgatcgac
      721 cgatacatca gcatccatag accactcgct tataagcgga ttgtaacacg cccaaaggcc
      781 gtggtcgcct tttgcttgat gtggacgatt gccatcgtca tcgccgtgct gccactgctc
      841 gggtggaact gcgaaaagtt gcagtccgtg tgcagtgaca tcttccccca tatcgacgag
      901 acctacctca tgttctggat tggcgtaaca agcgttctgt tgctgtttat tgtctacgct
      961 tacatgtaca ttctgtggaa ggcccatagt cacgcggtcc gcatgatcca gcgcggtacc
     1021 cagaagtcaa taattattca cacatctgaa gacggcaaag tacaggtcac aagaccggac
     1081 caggctcgga tggacatcag gcttgctaag acactggtgt tgatactggt ggtgctgatt
     1141 atctgctggg gacctctcct cgctattatg gtgtacgacg tgttcggcaa gatgaataag
     1201 ttgatcaaga ccgtatttgc cttctgttct atgctgtgcc tgcttaatag tactgtgaac
     1261 cccatcatct acgccctgcg ctctaaagac ttgcgccacg ccttccggtc catgtttccc
     1321 tcctgtgagg gtaccgcaca gcccctcgac aactccatgg gggatagcga ttgccttcac
     1381 aagcatgcaa acaacgcagc atccgtccac agggcggccg agagttgcat caagtctacc
     1441 gttaaaatcg ctaaagttac tatgtcagtc agcactgaca catctgccga agctctgatc
     1501 gataccggtg gacgcacccc acccagcctg ggtccccaag atgagtcctg caccaccgcc
     1561 agctcctccc tggccaagga cacttcatcg acmctcctcc ctggccaagg acacttcatc
     1621 gaccggtggc tctggaggtc tcgagatggg cgagagcctg ttcaagggac ctagggacta
     1681 caaccctatc agtagcacaa tttgtcacct gaccaacgag agtgatggcc acacaacaag
     1741 cctgtacggc atcgggttcg gaccctttat catcaccaac aagcacctgt tcaggcggaa
     1801 taatggcact ctgctggtgc agagcctgca cggggtgttc aaagtgaaga acacaaccac
     1861 tctgcagcag cacctgatcg atgggcggga tatgatcatc attaggatgc ccaaggactt
     1921 cccccctttt cctcagaaac tgaagttccg agagccccag agagaggaga gaatctgtct
     1981 ggtgaccaca aactttcagg agctcggtag tggtagtggg gagtttctgg caaccacact
     2041 ggaacggatc gagaaaaatt tcgtgattac tgatccgaga ctgcctgaca acccaatcat
     2101 ttttgcgagc gattccttcc tgcagctgac agaatattct cgggaagaga tcctggggcg
     2161 caattgccgt tttctgcagg gacccgagac agaccgtgcc actgttcgga aaatcagaga
     2221 tgctattgac aaccagactg aagtgaccgt tcagctgatc aattatacca agagcggcaa
     2281 gaagttctgg aacgtgttcc acctgcagcc gatgcgcgat tataagggcg acgtccagta
     2341 cttcattggc gtgcagctgg atggcaccga acgtcttcat ggcgccgctg agcgtgaggc
     2401 ggtctgcctg atcaaaaaga cagcctttca gattgctgag aacctgtact tccagggctc
     2461 tagattagat aaaagtaaag tgattaacag cgcattagag ctgcttaatg aggtcggaat
     2521 cgaaggttta acaacccgta aactcgccca gaagctaggt gtagagcagc ctacattgta
     2581 ttggcatgta aaaaataagc gggctttgct cgacgcctta gccattgaga tgttagatag
     2641 gcaccatact cacttttgcc ctttagaagg ggaaagctgg caagattttt tacgtaataa
     2701 cgctaaaagt tttagatgtg ctttactaag tcatcgcgat ggagcaaaag tacatttagg
     2761 tacacggcct acagaaaaac agtatgaaac tctcgaaaat caattagcct ttttatgcca
     2821 acaaggtttt tcactagaga atgcattata tgcactcagc gctgtggggc attttacttt
     2881 aggttgcgta ttggaagatc aagagcatca agtcgctaaa gaagaaaggg aaacacctac
     2941 tactgatagt atgccgccat tattacgaca agctatcgaa ttatttgatc accaaggtgc
     3001 agagccagcc ttcttattcg gccttgaatt gatcatatgc ggattagaaa aacaacttaa
     3061 atgtgaaagt gggtccgcgt acagccgcgc gcgtacgaaa aacaattacg ggtctaccat
     3121 cgagggcctg ctcgatctcc cggacgacga cgcccccgaa gaggcggggc tggcggctcc
     3181 gcgcctgtcc tttctccccg cgggacacac gcgcagactg tcgacggccc ccccgaccga
     3241 tgtcagcctg ggggacgagc tccacttaga cggcgaggac gtggcgatgg cgcatgccga
     3301 cgcgctagac gatttcgatc tggacatgtt gggggacggg gattccccgg gtccgggatt
     3361 taccccccac gactccgccc cctacggcgc tctggatatg gccgacttcg agtttgagca
     3421 gatgtttacc gatgcccttg gaattgacga gtacggtggg taggctagcc tccctatagt
     3481 gagtcgtatt acgtagatcc agacatgata agatacattg atgagtttgg acaaaccaca
     3541 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     3601 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     3661 caggttcagg gggaggtgtg ggaggttttt taattcgcgt ccgcggcgcc aatgcattgg
     3721 gcccggtacc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     3781 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3841 ccgggagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3901 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3961 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4021 ctgactcgct gcgctcggtc gttcggctgc gggagcggta tcagctcact caaaggcggt
     4081 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4141 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4201 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4261 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4321 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4381 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4441 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4501 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4561 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4621 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4681 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4741 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4801 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4861 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4921 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4981 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5041 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5101 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5161 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5221 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5281 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5341 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5401 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5461 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5521 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5581 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5641 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5701 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5761 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5821 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5881 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     5941 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     6001 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     6061 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     6121 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     6181 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     6241 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     6301 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     6361 gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg
     6421 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagt cgaccgccaa ttcaatatgg
     6481 cgtatatgga ctcatgccaa ttcaatatgg tggatctgga cctgtgccaa ttcaatatgg
     6541 cgtatatgga ctcgtgccaa ttcaatatgg tggatctgga ccccagccaa ttcaatatgg
     6601 cggacttggc accatgccaa ttcaatatgg cggacctggc actgtgccaa ctggggaggg
     6661 gtctacttgg cacggtgcca agtttgagga ggggtcttgg ccctgtgcca agtccgccat
     6721 attgaattgg catggtgcca ataatggcgg ccatattggc tatatgccag gatcaatata
     6781 taggcaatat ccaatatggc cctatgccaa tatggctatt ggccaggttc aatactatgt
     6841 attggcccta tgccatatag tattccatat atgggttttc ctattgacgt agatagcccc
     6901 tcccaatggg cggtcccata taccatatat ggggcttcct aataccgccc atagccactc
     6961 ccccattgac gtcaatggtc tctatatatg gtctttccta ttgacgtcat atgggcggtc
     7021 ctattgacgt atatggcgcc tcccccattg acgtcaatta cggtaaatgg cccgcctggc
     7081 tcaatgccca ttgacgtcaa taggaccacc caccattgac gtcaatggga tggctcattg
     7141 cccattcata tccgttctca cgccccctat tgacgtcaat gacggtaaat ggcccacttg
     7201 gcagtacatc aatatctatt aatagtaact tggcaagtac attactattg gaagtacgcc
     7261 agggtacatt ggcagtactc ccattgacgt caatggcggt aaatggcccg cgatggctgc
     7321 caagtacatc cccattgacg tcaatgggga ggggcaatga cgcaaatggg cgttccattg
     7381 acgtaaatgg gcggtaggcg tgcctaatgg gaggtctata taagcaatgc tcgtttaggg
     7441 aaccgccatt ctgcctgggg acgtcggagc aagcttgcca cc
//
