LOCUS       sequence-129521-        7921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7921)
  AUTHORS   Rose JC, Huang PS, Camp ND, Ye J, Leidal AM, Goreshnik I, Trevillian
            BM, Dickinson MS, Cunningham-Bryant D, Debnath J, Baker D, 
            Wolf-Yadlin A, Maly DJ
  TITLE     A computationally engineered RAS rheostat reveals RAS-ERK signaling 
            dynamics.
  JOURNAL   Nat Chem Biol. 2016 Nov 21. doi: 10.1038/nchembio.2244.
  PUBMED    27870838
REFERENCE   2  (bases 1 to 7921)
  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: "Nat Chem 
            Biol. 2016 Nov 21. doi: 10.1038/nchembio.2244."
COMMENT     Sequence Label: Braf
FEATURES             Location/Qualifiers
     source          1..7921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(63..80)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(234..822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(314..333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(993..1784,1785..1853))
                     /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     1616..1635
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1854..1958)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2033..2052)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2224..2603
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2604..2807
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2758..2778
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2852..2871
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2852..2870
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2927..2950
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(5332..5349)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5338..5562
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5608..6036
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5695..5714)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5905..5926
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6045..6065)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6050..6379
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6230..6365
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6292..6311
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6428..7453
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             6428..7453
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    7583..7704
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7620..7639)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7674..7693
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7753..7769)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7753..7769)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7766..7788)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7777..7793
                     /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(7801..7807,7808..7825,7826..7831))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7846..7867
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
       61 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
      121 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      181 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      241 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      301 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
      361 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
      421 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
      481 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
      541 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
      601 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
      661 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
      721 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
      781 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
      841 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
      901 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
      961 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1021 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1081 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     1141 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1201 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1261 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1321 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1381 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1441 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1501 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     1561 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     1621 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     1681 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     1741 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     1801 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     1861 ctttttcaat cttattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     1921 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     1981 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     2041 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     2101 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     2161 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     2221 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     2281 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     2341 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     2401 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     2461 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     2521 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     2581 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     2641 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     2701 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     2761 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     2821 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     2881 ctggctagcg tttaaactta agcttggtac cgagctcgga tccatggatt acaaggatga
     2941 cgatgacaag ggaattctgt acttccaatc caatgcggcg ctgagcggtg gcggtggtgg
     3001 cggcgcggag ccgggccagg ctctgttcaa cggggacatg gagcccgagg ccggcgccgg
     3061 cgccggcgcc gcggcctctt cggctgcgga ccctgccatt ccggaggagg tgtggaatat
     3121 caaacaaatg attaagttga cacaggaaca tatagaggcc ctattggaca aatttggtgg
     3181 ggagcataat ccaccatcaa tatatctgga ggcctatgaa gaatacacca gcaagctaga
     3241 tgcactccaa caaagagaac aacagttatt ggaatctctg gggaacggaa ctgatttttc
     3301 tgtttctagc tctgcatcaa tggataccgt tacatcttct tcctcttcta gcctttcagt
     3361 gctaccttca tctctttcag tttttcaaaa tcccacagat gtggcacgga gcaaccccaa
     3421 gtcaccacaa aaacctatcg ttagagtctt cctgcccaac aaacagagga cagtggtacc
     3481 tgcaaggtgt ggagttacag tccgagacag tctaaagaaa gcactgatga tgagaggtct
     3541 aatcccagag tgctgtgctg tttacagaat tcaggatgga gagaagaaac caattggttg
     3601 ggacactgat atttcctggc ttactggaga agaattgcat gtggaagtgt tggagaatgt
     3661 tccacttaca acacacaact ttgtacgaaa aacgtttttc accttagcat tttgtgactt
     3721 ttgtcgaaag ctgcttttcc agggtttccg ctgtcaaaca tgtggttata aatttcacca
     3781 gcgttgtagt acagaagttc cactgatgtg tgttaattat gaccaacttg atttgctgtt
     3841 tgtctccaag ttctttgaac accacccaat accacaggaa gaggcgtcct tagcagagac
     3901 tgccctaaca tctggatcat ccccttccgc acccgcctcg gactctattg ggccccaaat
     3961 tctcaccagt ccgtctcctt caaaatccat tccaattcca cagcccttcc gaccagcaga
     4021 tgaagatcat cgaaatcaat ttgggcaacg agaccgatcc tcatcagctc ccaatgtgca
     4081 tataaacaca atagaacctg tcaatattga tgacttgatt agagaccaag gatttcgtgg
     4141 tgatggagga tcaaccacag gtttgtctgc taccccccct gcctcattac ctggctcact
     4201 aactaacgtg aaagccttac agaaatctcc aggacctcag cgagaaagga agtcatcttc
     4261 atcctcagaa gacaggaatc gaatgaaaac acttggtaga cgggactcga gtgatgattg
     4321 ggagattcct gatgggcaga ttacagtggg acaaagaatt ggatctggat catttggaac
     4381 agtctacaag ggaaagtggc atggtgatgt ggcagtgaaa atgttgaatg tgacagcacc
     4441 tacacctcag cagttacaag ccttcaaaaa tgaagtagga gtactcagga aaacacgaca
     4501 tgtgaatatc ctactcttca tgggctattc cacaaagcca caactggcta ttgttaccca
     4561 gtggtgtgag ggctccagct tgtatcacca tctccatatc attgagacca aatttgagat
     4621 gatcaaactt atagatattg cacgacagac tgcacagggc atggattact tacacgccaa
     4681 gtcaatcatc cacagagacc tcaagagtaa taatatattt cttcatgaag acctcacagt
     4741 aaaaataggt gattttggtc tagctacagt gaaatctcga tggagtgggt cccatcagtt
     4801 tgaacagttg tctggatcca ttttgtggat ggcaccagaa gtcatcagaa tgcaagataa
     4861 aaatccatac agctttcagt cagatgtata tgcatttgga attgttctgt atgaattgat
     4921 gactggacag ttaccttatt caaacatcaa caacagggac cagataattt ttatggtggg
     4981 acgaggatac ctgtctccag atctcagtaa ggtacggagt aactgtccaa aagccatgaa
     5041 gagattaatg gcagagtgcc tcaaaaagaa aagagatgag agaccactct ttccccaaat
     5101 tctcgcctct attgagctgc tggcccgctc attgccaaaa attcaccgca gtgcatcaga
     5161 accctccttg aatcgggctg gtttccaaac agaggatttt agtctatatg cttgtgcttc
     5221 tccaaaaaca cccatccagg cagggggata tggtgcgttt cctgtccact gacattggaa
     5281 gtggataact cgagcatgca tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     5341 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     5401 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     5461 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     5521 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     5581 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     5641 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     5701 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     5761 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     5821 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5881 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     5941 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     6001 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     6061 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     6121 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     6181 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     6241 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     6301 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     6361 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     6421 gcacgtgatg aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa
     6481 gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag
     6541 cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta
     6601 caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct
     6661 tgacattggg gaatttagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt
     6721 cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc
     6781 catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc
     6841 gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca
     6901 tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct
     6961 cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga
     7021 tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag
     7081 cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg
     7141 gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg
     7201 atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt
     7261 ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg
     7321 atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac
     7381 cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag
     7441 ggcaaaggaa tagcacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt
     7501 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     7561 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     7621 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     7681 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     7741 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     7801 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     7861 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     7921 g
//
