LOCUS       sequence-451903-        8642 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Used to integrate 3xFlag-tdMCP-BRaf and MS2-circRNA barcodes into 
            RNA-Protein Landing Pad cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8642)
  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 8642)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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 with MS2-circRNA
FEATURES             Location/Qualifiers
     source          1..8642
                     /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    427..464
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      510..519
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             519..584
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    join(3704..4278,4279..4281,4282..4290)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3871..3888)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4098..4117
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(4436..4454)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4704..4723
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4987..5043
                     /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"
     polyA_signal    5740..5861
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5777..5796)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5831..5850
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(5870..5888)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5909..5925)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5909..5925)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5922..5944)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5933..5949
                     /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(5957..5963,5964..5981,5982..5987))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6002..6023
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6140..6157)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6311..6899)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6391..6410)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7070..7861,7862..7930))
                     /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     7693..7712
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7931..8035)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8061..8516)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8198..8219)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8410..8429
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca ctatagggcg
       61 aattgggtac cgggcccccc cgcatgcact agtaaaaaaa gcggaccgaa gtccgctcta
      121 gagcgtggac tgtaccctcc acttttatcc gggcttggga ccggcattgg cagtgttcta
      181 cagtccacgc cgaccgcggc cacggctctg atgcgtctgg attacgactc gtcgtcacta
      241 gatgtacata tttttgaatt cgcgtgtaca acgtccggag cccacactct actcgacacg
      301 taccctgatg gtgtacgtgg acccgaccgt ctccggacgt gcggccgcac tcggcatcgt
      361 taggcggttt cccgccccct cccattttat ccgggcttgg gaccggcgag tgcggcccgt
      421 cgacccggct tgtcgacgac ggcggtctcc gtcgtcagga tcatccgccg cggcttgagc
      481 ggactctagc gtttaaactt aagcttgccg ccaccatgga ttataaagat cacgacggtg
      541 actataagga tcacgatatt gactacaagg atgacgacga caaaggttca ggaagcggtt
      601 ctggagcctc caattttact cagttcgtct tggtggataa cggcggtacg ggggacgtta
      661 cggtcgctcc ctccaatttt gccaacggaa tagccgagtg gatttccagt aattccaggt
      721 cacaagcgta taaagtaacg tgttccgtca gacaaagctc cgcccaaaat cgaaagtata
      781 ccatcaaagt agaagttcca aagggtgcgt ggagatctta tctgaacatg gaactcacta
      841 ttcccatctt cgccaccaac tccgactgtg aacttatcgt taaggcaatg caaggtctgc
      901 tgaaagatgg aaaccctatc cctagcgcca tcgccgcaaa ctccggcatc tatgccatgg
      961 cttcaaactt cacccagttt gtgcttgtag acaatggtgg cacgggcgac gtgacagtgg
     1021 caccttccaa ctttgctaac ggcattgcag aatggattag ttctaattcc cgctcccagg
     1081 cttataaagt tacgtgtagt gttcgccagt caagcgcaca aaaccgaaag tacaccataa
     1141 aggttgaagt ccccaaggga gcctggcgat cataccttaa catggagctt actattccga
     1201 tcttcgctac caattcagat tgtgaattga ttgtcaaagc tatgcaaggc ctgctcaagg
     1261 atggtaaccc gatacctagc gctatcgccg ccaattcagg catttatggc agtggaggtt
     1321 ccggatccgg atctggtggc gctgcgctga gcggtggagg tggaggcggt gcagaacccg
     1381 ggcaagcatt gttcaatggg gatatggaac ctgaagctgg cgcaggagcc ggagccgcgg
     1441 cctcttcggc tgccgatcct gctattcctg aggaggtgtg gaacatcaaa cagatgatta
     1501 aactgacgca agagcatatt gaagccctgc ttgataagtt cgggggcgag cataaccccc
     1561 ccagcattta tctggaggca tatgaggaat atacgtctaa actcgacgcc cttcaacagc
     1621 gggaacagca gctccttgaa agcttgggca atgggacgga tttctcagtt agttccagtg
     1681 ccagtatgga tacagtgacc tcatcatcca gttcctccct gtctgtcctg ccttcttctc
     1741 tctctgtttt ccaaaacccg actgatgtag caaggtctaa tcctaaaagc ccacaaaagc
     1801 cgattgttcg ggtattcctg ccaaacaaac aaagaacagt tgtcccagcg cgatgcggcg
     1861 tcactgttcg agatagtctc aaaaaagcac tgatgatgcg gggattgatt cccgaatgct
     1921 gcgcagttta ccggattcag gatggggaga aaaaaccaat aggttgggac acggatataa
     1981 gttggcttac aggggaggag cttcacgttg aggttctgga gaatgttccc ttgacaacac
     2041 ataattttgt cagaaagacg ttcttcactt tggctttctg tgacttttgt agaaagctgc
     2101 tcttccaggg ctttcgatgt ctgacctgcg ggtataaatt ccaccagcgg tgctccacag
     2161 aggtaccttt gatgtgtgtc aactatgatc aacttgacct tcttttcgtc agtaagttct
     2221 ttgaacacca tccgatccct caggaggaag cctcccttgc cgagactgcc ttgaccagtg
     2281 gctcctctcc ttctgcacct gcatccgaca gtataggccc gcaaatattg acctcaccga
     2341 gccccagtaa atctattccg atcccgcagc cctttcgccc tgcggacgaa gatcatagga
     2401 atcagtttgg acagcgcgac agatcctcca gtgcgccgaa cgtacacata aatacaattg
     2461 aacctgttaa tatagacgat ttgattcgcg accaagggtt taggggtgac ggagggtcaa
     2521 ccaccggttt gtctgctact ccaccagctt ctctgccagg gtctctcaca aatgttaaag
     2581 cattgcaaaa atcccctgga cctcaacgag aaaggaagag cagctcatct agcgaggacc
     2641 gcaacagaat gaagactctc ggtagaagag actcgagtga cgactgggaa ataccagacg
     2701 gtcaaatcac ggtcggtcag cggatcggat caggctcctt cgggactgta tataaaggta
     2761 aatggcacgg cgacgttgcg gtcaaaatgc tgaacgttac agcaccaacc ccccaacagc
     2821 ttcaagcgtt caagaacgaa gtcggggtac tgcgcaaaac tcggcatgtc aatatattgc
     2881 tgttcatggg ttactcaacc aagcctcaac tcgccatagt tacccagtgg tgtgaaggca
     2941 gctcacttta ccatcacctg cacataatag agaccaagtt cgagatgatc aaactcattg
     3001 acattgcgcg gcaaacagcg caggggatgg actatctgca tgcaaagtct attatccata
     3061 gagacctcaa gtcaaacaac atattcttgc acgaggatct caccgttaaa attggagact
     3121 ttggtctcgc aactgttaag tcacggtgga gtgggtcaca tcagttcgag cagctgtccg
     3181 gcagcatact gtggatggct ccagaggtca ttcgcatgca ggataagaac ccttattctt
     3241 tccaatccga tgtttatgca tttggcatcg tcctctatga gctgatgacc ggacaactcc
     3301 cttacagcaa catcaacaat cgagaccaga tcatcttcat ggtcgggcga ggatacctca
     3361 gccccgatct ctcaaaggtt cgatcaaatt gccctaaagc gatgaaacgg cttatggcgg
     3421 agtgtttgaa aaagaaacgc gacgaacgcc ctttgttccc tcaaatcttg gcatcaatcg
     3481 agttgctggc tagaagtctt ccaaaaatac acagaagcgc atccgagcca agcctcaatc
     3541 gggcaggatt ccagaccgag gatttctctc tttatgcctg cgcatctcca aagacaccca
     3601 tacaggccgg cgggtacggc gcctttcctg tgcactgaga gctcgctgac tccgctagcg
     3661 ccggtcttaa ctgatcactg acgcgttgga atcagagaga ctgcccctct ccctctcccc
     3721 cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt
     3781 tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct
     3841 tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga
     3901 atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga
     3961 ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac
     4021 gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag
     4081 ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc
     4141 agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg
     4201 tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt
     4261 gaaaaacacg atgataatat ggccacaacc atggtgagca agggcgagga ggataacatg
     4321 gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac
     4381 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     4441 ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc
     4501 atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg
     4561 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
     4621 accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc
     4681 ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
     4741 tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg
     4801 aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag
     4861 cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac
     4921 gaggactata ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc
     4981 agcggagcta ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga
     5041 cctaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     5101 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     5161 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     5221 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     5281 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     5341 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     5401 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accaaggaaa gggattggga
     5461 tcagcagttg ttctccctgg agtcgaggca gctgaacggg ctggcgtccc tgcttttctc
     5521 gaaacctctg ctcctcgcaa tctccccttt tatgagcgcc ttggatttac tgtcaccgcc
     5581 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagta ataggatcat
     5641 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     5701 cctgaacctg aaacataaaa tgaatgcatt gttgttgtta acttgtttat tgcagcttat
     5761 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5821 cattctagtt gtggtttgtc caaactcatc aatgtatctt aggagatccc ctttagtgag
     5881 ggttaatttc gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5941 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     6001 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     6061 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6121 ttgggcgctc ttccgcttcc tcgctcactg actcgctgca ctcggtcgtt cggctgcggc
     6181 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6241 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6301 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6361 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6421 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6481 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6541 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6601 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6661 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6721 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6781 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6841 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6901 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6961 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     7021 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     7081 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     7141 tccccgtcat gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     7201 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     7261 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7321 gttgtcggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7381 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7441 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7501 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7561 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7621 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7681 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7741 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7801 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7861 gaacaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7921 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7981 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     8041 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     8101 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     8161 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     8221 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     8281 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     8341 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     8401 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     8461 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     8521 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     8581 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     8641 tt
//
