LOCUS       sequence-213484-        4599 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 4599)
  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 4599)
  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..4599
                     /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(2411..2431)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2411..2429)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2450..2466)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2450..2466)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2463..2485)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2474..2490
                     /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(2498..2504,2505..2522,2523..2528))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2543..2564
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2681..2698)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2852..3440)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2932..2951)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3611..4402,4403..4471))
                     /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     4234..4253
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4472..4576)
                     /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 tcactgccca
     2401 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     2461 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     2521 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     2581 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     2641 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2701 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2761 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2821 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2881 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2941 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3001 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3061 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3121 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3181 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3241 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     3301 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3361 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3421 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3481 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3541 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3601 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3661 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3721 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3781 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3841 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3901 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3961 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4021 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4081 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4141 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4201 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4261 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4321 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4381 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4441 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4501 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4561 aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
