LOCUS       sequence-451902-        7904 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to integrate HA-tagged 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 7904)
  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 7904)
  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) BRaf with MS2-circRNA
FEATURES             Location/Qualifiers
     source          1..7904
                     /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)"
     CDS             519..545
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             555..581
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(2966..3540,3541..3543,3544..3552)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3133..3150)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3360..3379
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(3698..3716)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3966..3985
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4249..4305
                     /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    5002..5123
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5039..5058)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5093..5112
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(5132..5150)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5171..5187)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5171..5187)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5184..5206)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5195..5211
                     /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(5219..5225,5226..5243,5244..5249))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5264..5285
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5402..5419)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5573..6161)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5653..5672)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6332..7123,7124..7192))
                     /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     6955..6974
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7193..7297)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7323..7778)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7460..7481)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7672..7691
                     /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 ccaccatgta tccctatgac gtgccggact
      541 acgctggctc cggctatccc tatgatgtgc cggattacgc aggttcagga agcggttctg
      601 gagctgcgct gagcggtgga ggtggaggcg gtgcagaacc cgggcaagca ttgttcaatg
      661 gggatatgga acctgaagct ggcgcaggag ccggagccgc ggcctcttcg gctgccgatc
      721 ctgctattcc tgaggaggtg tggaacatca aacagatgat taaactgacg caagagcata
      781 ttgaagccct gcttgataag ttcgggggcg agcataaccc ccccagcatt tatctggagg
      841 catatgagga atatacgtct aaactcgacg cccttcaaca gcgggaacag cagctccttg
      901 aaagcctggg caatgggacg gatttctcag ttagttccag tgccagtatg gatacagtga
      961 cctcatcatc cagttcctcc ctgtctgtcc tgccttcttc tctctctgtt ttccaaaacc
     1021 cgactgatgt agcaaggtct aatcctaaaa gcccacaaaa gccgattgtt cgggtattcc
     1081 tgccaaacaa acaaagaaca gttgtcccag cgcgatgcgg cgtcactgtt cgagatagtc
     1141 tcaaaaaagc actgatgatg cggggattga ttcccgaatg ctgcgcagtt taccggattc
     1201 aggatgggga gaaaaaacca ataggttggg acacggatat aagttggctt acaggggagg
     1261 agcttcacgt tgaggttctg gagaatgttc ccttgacaac acataatttt gtcagaaaga
     1321 cgttcttcac tttggctttc tgtgactttt gtagaaagct gctcttccag ggctttcgat
     1381 gtcagacctg cgggtataaa ttccaccagc ggtgctccac agaggtacct ttgatgtgtg
     1441 tcaactatga tcaacttgac cttcttttcg tcagtaagtt ctttgaacac catccgatcc
     1501 ctcaggagga agcctccctt gccgagactg ccttgaccag tggctcctct ccttctgcac
     1561 ctgcatccga cagtataggc ccgcaaatat tgacctcacc gagccccagt aaatctattc
     1621 cgatcccgca gccctttcgc cctgcggacg aagatcatag gaatcagttt ggacagcgcg
     1681 acagatcctc cagtgcgccg aacgtacaca taaatacaat tgaacctgtt aatatagacg
     1741 atttgattcg cgaccaaggg tttaggggtg acggagggtc aaccaccggt ttgtctgcta
     1801 ctccaccagc ttctctgcca gggtctctca caaatgttaa agcattgcaa aaatcccctg
     1861 gacctcaacg agaaaggaag agcagctcat ctagcgagga ccgcaacaga atgaagactc
     1921 tcggtagaag agactcgagt gacgactggg aaataccaga cggtcaaatc acggtcggtc
     1981 agcggatcgg atcaggctcc ttcgggactg tatataaagg taaatggcac ggcgacgttg
     2041 cggtcaaaat gctgaacgtt acagcaccaa ccccccaaca gcttcaagcg ttcaagaacg
     2101 aagtcggggt actgcgcaaa actcggcatg tcaatatatt gctgttcatg ggttactcaa
     2161 ccaagcctca actcgccata gttacccagt ggtgtgaagg cagctcactt taccatcacc
     2221 tgcacataat agagaccaag ttcgagatga tcaaactcat tgacattgcg cggcaaacag
     2281 cgcaggggat ggactatctg catgcaaagt ctattatcca tagagacctc aagtcaaaca
     2341 acatattctt gcacgaggat ctcaccgtta aaattggaga ctttggtctc gcaactgtta
     2401 agtcacggtg gagtgggtca catcagttcg agcagctgtc cggcagcata ctgtggatgg
     2461 ctccagaggt cattcgcatg caggataaga acccttattc tttccaatcc gatgtttatg
     2521 catttggcat cgtcctctat gagctgatga ccggacaact cccttacagc aacatcaaca
     2581 atcgagacca gatcatcttc atggtcgggc gaggatacct cagccccgat ctctcaaagg
     2641 ttcgatcaaa ttgccctaaa gcgatgaaac ggcttatggc ggagtgtttg aaaaagaaac
     2701 gcgacgaacg ccctttgttc cctcaaatct tggcatcaat cgagttgctg gctagaagtc
     2761 ttccaaaaat acacagaagc gcatccgagc caagcctcaa tcgggcagga ttccagaccg
     2821 aggatttctc tctttatgcc tgcgcatctc caaagacacc catacaggcc ggcgggtacg
     2881 gcgcctttcc tgtgcactga gagctcgctg actccgctag cgccggtctt aactgatcac
     2941 tgacgcgttg gaatcagaga gactgcccct ctccctctcc cccccctaac gttactggcc
     3001 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc
     3061 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta
     3121 ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag
     3181 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
     3241 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg
     3301 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat
     3361 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta
     3421 tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa
     3481 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat
     3541 atggccacaa ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc
     3601 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
     3661 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
     3721 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
     3781 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
     3841 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
     3901 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     3961 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     4021 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     4081 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     4141 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta taccatcgtg
     4201 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcagcggagc tactaacttc
     4261 agcctgctga agcaggctgg agacgtggag gagaaccctg gacctaccga gtacaagccc
     4321 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     4381 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     4441 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     4501 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     4561 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     4621 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     4681 accgtcggcg tctcgcccga ccaccaagga aagggattgg gatcagcagt tgttctccct
     4741 ggagtcgagg cagctgaacg ggctggcgtc cctgcttttc tcgaaacctc tgctcctcgc
     4801 aatctcccct tttatgagcg ccttggattt actgtcaccg ccgacgtcga ggtgcccgaa
     4861 ggaccgcgca cctggtgcat gacccgcaag taataggatc ataatcagcc ataccacatt
     4921 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     4981 aatgaatgca ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     5041 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5101 tccaaactca tcaatgtatc ttaggagatc ccctttagtg agggttaatt tcgagcttgg
     5161 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5221 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5281 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5341 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5401 cctcgctcac tgactcgctg cactcggtcg ttcggctgcg gcgagcggta tcagctcact
     5461 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5521 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5581 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5641 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5701 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5761 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5821 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5881 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5941 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6001 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6061 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6121 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6181 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6241 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6301 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6361 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc atgtagataa
     6421 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6481 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6541 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgtcgg gaagctagag
     6601 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6661 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6721 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6781 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6841 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6901 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6961 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7021 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7081 actgatcttc agcatctttt actttcacca gcgtttctgg gtgaacaaaa acaggaaggc
     7141 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7201 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7261 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7321 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     7381 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     7441 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     7501 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     7561 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     7621 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     7681 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     7741 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
     7801 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     7861 gggatgtgct gcaaggcgat taagttgggt aacgccaggg tttt
//
