LOCUS       sequence-197141-        4598 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CLIPf under the TRE promoter in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4598)
  AUTHORS   Sakaguchi R, Leiwe MN, Imai T
  TITLE     Bright multicolor labeling of neuronal circuits with fluorescent 
            proteins and chemical tags.
  JOURNAL   eLife. 2018 Nov 20;7. pii: 40350. doi: 10.7554/eLife.40350.
  PUBMED    30454553
REFERENCE   2  (bases 1 to 4598)
  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: "eLife."; 
            date: "2018-11-20"; volume: "7. pii: 40350. doi"; pages: " 
            10.7554/eLife.40350"
COMMENT     Sequence Label: pBS-TRE-CLIPf-WPRE
FEATURES             Location/Qualifiers
     source          1..4598
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        669..983
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    677..695
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    713..731
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    748..766
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    784..802
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    855..873
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    891..909
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     956..980
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      994..1003
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1000..1545
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylcytosine 
                     derivatives"
                     /label=CLIP-tag(TM)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLIQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISESHLAALVGNPAATAAVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLA
                     VKEWLLAHEGHRLGKPGLG"
     misc_feature    1567..2155
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1620..1640)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2038..2049)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2316..2397
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2353..2372)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2410..2430)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2410..2428)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2449..2465)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2449..2465)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2462..2484)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2473..2489
                     /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(2497..2503,2504..2521,2522..2527))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2542..2563
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2680..2697)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2851..3439)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2931..2950)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3610..4401,4402..4470))
                     /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     4233..4252
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4471..4575)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggctttcgt
      661 cttcactcga gtttactccc tatcagtgat agagaacgta tgtcgagttt actccctatc
      721 agtgatagag aacgatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgagtt
      781 tactccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac
      841 gtatgtcgag tttatcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag
      901 tgatagagaa cgtatgtcga ggtaggcgtg tacggtggga ggcctatata agcagagctc
      961 gtttagtgaa ccgtcagatc gcctggagaa ttcgccacca tggacaaaga ctgcgaaatg
     1021 aagcgcacca ccctggatag ccctctgggc aagctggaac tgtctgggtg cgaacagggc
     1081 ctgcaccgta tcatcttcct gggcaaagga acatctgccg ccgacgccgt ggaagtgcct
     1141 gccccagccg ccgtgctggg cggaccagag ccactgatcc aggccaccgc ctggctcaac
     1201 gcctactttc accagcctga ggccatcgag gagttccctg tgccagccct gcaccaccca
     1261 gtgttccagc aggagagctt tacccgccag gtgctgtgga aactgctgaa agtggtgaag
     1321 ttcggagagg tcatcagcga gagccacctg gccgccctgg tgggcaatcc cgccgccacc
     1381 gccgccgtga acaccgccct ggacggaaat cccgtgccca ttctgatccc ctgccaccgg
     1441 gtggtgcagg gcgacagcga cgtggggccc tacctgggcg ggctcgccgt gaaagagtgg
     1501 ctgctggccc acgagggcca cagactgggc aagcctgggc tgggttaggc ggccgcgctt
     1561 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     1621 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     1681 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     1741 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     1801 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     1861 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     1921 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     1981 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     2041 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     2101 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     2161 taccgtcgac gcggccgcat cgataagctt gtcgacgata tctctagagg atcataatca
     2221 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     2281 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     2341 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgccag
     2401 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt
     2461 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     2521 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     2581 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     2641 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     2701 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     2761 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     2821 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2881 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2941 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3001 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3061 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3121 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3181 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3241 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     3301 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3361 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3421 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3481 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3541 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3601 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3661 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3721 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     3781 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3841 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3901 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3961 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4021 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4081 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4141 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4201 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4261 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4321 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4381 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4441 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4501 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4561 aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
