LOCUS       sequence-451900-        8329 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to integrate 3xFlag-tdMCP-BRaf without MS2-circRNA barcdes into
            landing pad cell lines containing a single promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8329)
  AUTHORS   Simon JJ, Fowler DM, Maly DJ
  TITLE     Multiplexed profiling of intracellular protein abundance, activity, 
            interactions and druggability with LABEL-seq.
  JOURNAL   Nat Methods. 2024 Nov;21(11):2094-2106. doi: 
            10.1038/s41592-024-02456-7. Epub 2024 Oct 21.
  PUBMED    39433876
REFERENCE   2  (bases 1 to 8329)
  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/s41592-024-02456-7"; journalName: "Nat Methods"; date: 
            "2024-11"; volume: "21"; issue: "11"; pages: "2094-2106"
COMMENT     Sequence Label: (attB) tdMCP-tagged BRaf
FEATURES             Location/Qualifiers
     source          1..8329
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..23
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     15..32
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     16..32
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     39..58
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        39..57
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    97..134
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      151..160
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             160..225
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    join(3383..3957,3958..3960,3961..3969)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3550..3567)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3777..3796
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(4115..4133)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4383..4402
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4666..4722
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     5216..5236
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5427..5548
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5464..5483)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5518..5537
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(5557..5575)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5596..5612)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5596..5612)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5609..5631)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5620..5636
                     /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(5644..5650,5651..5668,5669..5674))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5689..5710
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5827..5844)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5998..6586)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6078..6097)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6757..7548,7549..7617))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFVHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYMTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7380..7399
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7618..7722)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7748..8203)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7885..7906)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8097..8116
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca ctatagggcg
       61 aattgggtac cgggcccccc cgcatgcact agtcccggct tgtcgacgac ggcggtctcc
      121 gtcgtcagga tcatcgccct taagcttgcc gccaccatgg attataaaga tcatgacggg
      181 gactataagg atcacgatat tgactacaag gatgacgacg acaaaggttc tggcagtggc
      241 agcggagctt ctaactttac tcagttcgtt ctcgtcgaca atggcggaac tggcgacgtg
      301 actgtcgccc caagcaactt cgctaacggg atcgctgaat ggatcagctc taactcgcgt
      361 tcacaggctt acaaagtaac ctgtagcgtt cgtcagagct ctgcgcagaa tcgcaaatac
      421 accatcaaag tcgaggtgcc taaaggcgcc tggcgttcgt acttaaatat ggaactaacc
      481 attccaattt tcgccacgaa ttccgactgc gagcttattg ttaaggcaat gcaaggtctc
      541 ctaaaagatg gaaacccgat tccctcagca atcgcagcaa actccggcat ctacgcaatg
      601 gcttctaact ttactcagtt cgttctcgtc gacaatggcg gaactggcga cgtgactgtc
      661 gccccaagca acttcgctaa cgggatcgct gaatggatca gctctaactc gcgttcacag
      721 gcttacaaag taacctgtag cgttcgtcag agctctgcgc agaatcgcaa atacaccatc
      781 aaagtcgagg tgcctaaagg cgcctggcgt tcgtacttaa atatggaact aaccattcca
      841 attttcgcca cgaattccga ctgcgagctt attgttaagg caatgcaagg tctcctaaaa
      901 gatggaaacc cgattccctc agcaatcgca gcaaactccg gcatctacgg ctccggaggc
      961 tctggtagcg gatctggtgg cgcggcgctg agcggtggcg gtggtggcgg cgcggagccg
     1021 ggccaggctc tgttcaacgg ggacatggag cccgaggccg gcgccggcgc cggcgccgcg
     1081 gcctcttcgg ctgcggaccc tgccattccg gaggaggtgt ggaatatcaa acaaatgatt
     1141 aagttgacac aggaacatat agaggcccta ttggacaaat ttggtgggga gcataatcca
     1201 ccatcaatat atctggaggc ctatgaagaa tacaccagca agctagatgc actccaacaa
     1261 agagaacaac agttattgga atctctgggg aacggaactg atttttctgt ttctagctct
     1321 gcatcaatgg ataccgttac atcttcttcc tcttctagcc tttcagtgct accttcatct
     1381 ctttcagttt ttcaaaatcc cacagatgtg gcacggagca accccaagtc accacaaaaa
     1441 cctatcgtta gagtcttcct gcccaacaaa cagaggacag tggtacctgc aaggtgtgga
     1501 gttacagtcc gagacagtct aaagaaagca ctgatgatga gaggtctaat cccagagtgc
     1561 tgtgctgttt acagaattca ggatggagag aagaaaccaa ttggttggga cactgatatt
     1621 tcctggctta ctggagaaga attgcatgtg gaagtgttgg agaatgttcc acttacaaca
     1681 cacaactttg tacgaaaaac gtttttcacc ttagcatttt gtgacttttg tcgaaagctg
     1741 cttttccagg gtttccgctg tcaaacatgt ggttataaat ttcaccagcg ttgtagtaca
     1801 gaagttccac tgatgtgtgt taattatgac caacttgatt tgctgtttgt ctccaagttc
     1861 tttgaacacc acccaatacc acaggaagag gcgtccttag cagagactgc cctaacatct
     1921 ggatcatccc cttccgcacc cgcctcggac tctattgggc cccaaattct caccagtccg
     1981 tctccttcaa aatccattcc aattccacag cccttccgac cagcagatga agatcatcga
     2041 aatcaatttg ggcaacgaga ccgatcctca tcagctccca atgtgcatat aaacacaata
     2101 gaacctgtca atattgatga cttgattaga gaccaaggat ttcgtggtga tggaggatca
     2161 accacaggtt tgtctgctac cccccctgcc tcattacctg gctcactaac taacgtgaaa
     2221 gccttacaga aatctccagg acctcagcga gaaaggaagt catcttcatc ctcagaagac
     2281 aggaatcgaa tgaaaacact tggtagacgg gactcgagtg atgattggga gattcctgat
     2341 gggcagatta cagtgggaca aagaattgga tctggatcat ttggaacagt ctacaaggga
     2401 aagtggcatg gtgatgtggc agtgaaaatg ttgaatgtga cagcacctac acctcagcag
     2461 ttacaagcct tcaaaaatga agtaggagta ctcaggaaaa cacgacatgt gaatatccta
     2521 ctcttcatgg gctattccac aaagccacaa ctggctattg ttacccagtg gtgtgagggc
     2581 tccagcttgt atcaccatct ccatatcatt gagaccaaat ttgagatgat caaacttata
     2641 gatattgcac gacagactgc acagggcatg gattacttac acgccaagtc aatcatccac
     2701 agagacctca agagtaataa tatatttctt catgaagacc tcacagtaaa aataggtgat
     2761 tttggtctag ctacagtgaa atctcgatgg agtgggtccc atcagtttga acagttgtct
     2821 ggatccattt tgtggatggc accagaagtc atcagaatgc aagataaaaa tccatacagc
     2881 tttcagtcag atgtatatgc atttggaatt gttctgtatg aattgatgac tggacagtta
     2941 ccttattcaa acatcaacaa cagggaccag ataattttta tggtgggacg aggatacctg
     3001 tctccagatc tcagtaaggt acggagtaac tgtccaaaag ccatgaagag attaatggca
     3061 gagtgcctca aaaagaaaag agatgagaga ccactctttc cccaaattct cgcctctatt
     3121 gagctgctgg cccgctcatt gccaaaaatt caccgcagtg catcagaacc ctccttgaat
     3181 cgggctggtt tccaaacaga ggattttagt ctatatgctt gtgcttctcc aaaaacaccc
     3241 atccaggcag ggggatatgg tgcgtttcct gtccactgac attggaagtg gataactcga
     3301 gcatgcatct agagggcccg tttaaacgct agcgggatcc accggatcta gataactgat
     3361 cactgtggaa tcagagagac tgcccctctc cctctccccc ccctaacgtt actggccgaa
     3421 gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt
     3481 cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg
     3541 gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc
     3601 ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc
     3661 ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa
     3721 aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc
     3781 tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg
     3841 gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac
     3901 gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataatatg
     3961 gccacaacca tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
     4021 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
     4081 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     4141 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
     4201 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
     4261 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     4321 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     4381 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
     4441 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     4501 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     4561 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     4621 cagtacgaac gcgccgaggg ccgccactcc accggcggca gcggagctac taacttcagc
     4681 ctgctgaagc aggctggaga cgtggaggag aaccctggac ctaccgagta caagcccacg
     4741 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     4801 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     4861 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     4921 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     4981 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     5041 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     5101 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     5161 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     5221 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     5281 ccgcgcacct ggtgcatgac ccgcaagtaa tagggatcct gatcataatc agccatacca
     5341 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     5401 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     5461 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5521 gtttgtccaa actcatcaat gtatcttagg agatcccctt tagtgagggt taatttcgag
     5581 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     5641 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5701 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5761 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5821 cgcttcctcg ctcactgact cgctgcactc ggtcgttcgg ctgcggcgag cggtatcagc
     5881 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5941 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     6001 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     6061 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6121 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6181 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6241 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6301 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6361 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6421 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6481 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6541 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6601 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6661 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6721 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6781 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcatgta
     6841 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6901 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6961 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gtcgggaagc
     7021 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     7081 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7141 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7201 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7261 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7321 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     7381 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7441 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7501 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgaa caaaaacagg
     7561 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7621 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7681 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7741 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     7801 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     7861 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     7921 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     7981 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     8041 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     8101 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     8161 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg
     8221 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     8281 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggtttt
//
