LOCUS       sequence-171532-        7339 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses CIAR in mammalian cells. Used to generate Flp-In stables..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7339)
  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 7339)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: CIAR_pcDNA5/FRT/TO
FEATURES             Location/Qualifiers
     source          1..7339
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     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    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(3291..3308)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3297..3521
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3654..3673)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3805..3852
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3860..4882
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3860..4882
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5012..5133
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5049..5068)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5103..5122
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5182..5198)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5182..5198)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5195..5217)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5206..5222
                     /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(5230..5236,5237..5254,5255..5260))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5275..5296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5413..5430)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5584..6172)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5664..5683)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6343..7134,7135..7203))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRHIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6966..6985
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7204..7308)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacatggc tcctcctaat ctgtgggcgg cacagcgtta tgggcgtgaa
     1021 ctgcggcgta tggccgatga gggtgagggt tccttcaagg gtagcggaac tgggtctggg
     1081 actgggagtg gcaccggttc agggacagga gatgtgtatc gctttgctga gcctgactcc
     1141 gaagagaaca ttatcttcga agagaatatg cagcctaaag ctggtattcc gattatcaaa
     1201 gccgggaccg tgattaagct gatcgagcgt ctgacatatc acatgtacgc cgaccccaac
     1261 tttgttcgca ccttcctgac cacatatcgt tccttttgca agccacagga actgctgtct
     1321 ctgattatcg agcggttcga aattccagaa ccagagccaa ccgaggcaga tcgtattgcg
     1381 atcgaaaacg gcgaccagcc tctgagcgcg gagctgaaac gttttcggaa ggaatacatc
     1441 cagccggtcc agctgcgggt tctgaatgtc tgtcgccatt gggtggagca ccatttttat
     1501 gatttcgaac gggacgctta cctgctgcag cgcatggaag agttcattgg tacagtccgc
     1561 gggaaagcca tgaaaaagtg ggtggaatcc attactaaga ttatccagcg caaaaagatc
     1621 gcccgtgata acgggccagg acacaatatc acatttcaga gctccccgcc cactgtggag
     1681 tggcacattt ctcgtcccgg tcatatcgaa accttcgatc tgctgacact gcatccaatt
     1741 gagatcgctc gtcagctgac actgctggaa tctgacctgt atcgtgcagt gcagccaagc
     1801 gagctggtgg gctccgtttg gaccaaagag gataaggaaa ttaacagtcc aaatctgctg
     1861 aaaatgatcc gccacactac caacctgact ctgtggtttg aaaagtgcat tgttgaaacc
     1921 gagaatctgg aagagcgtgt cgcggtggtt agccggatta tcgagatcct gcaggtgttt
     1981 caggaactga acaatttcaa cggcgttctg gaggtcgtgt ccgcaatgaa cagcagccct
     2041 gtgtatcgcc tggaccatac cttcgaacag attccgagcc gtcagaaaaa gatcctggaa
     2101 gaggcgcacg agctgtccga agatcattat aaaaagtacc tggcaaaact gcgctctatt
     2161 aacccacctt gcgtgccgtt tttcggtatt tacctgacta acatcctgaa gaccgaagag
     2221 ggaaatccgg aggttctgaa acgtcacggc aaggaactga tcaacttttc aaaacgccgt
     2281 aaggtcgccg agattctcgg tgaaatccag cagtatcaga atcagcctta ctgtctgcgt
     2341 gtggagagcg acattaaacg gtttttcgaa aacctgaatc cgatggggaa cagtatggaa
     2401 aaagagttta ccgattacct gttcaataag tcactggaaa tcgagcctgg ctctggttca
     2461 cagagcaacc gtgaactggt tgtcgatttt ctgtcctata aactgtctca gaagggctac
     2521 tcctggtctc agttcagcga cgtcgaagag aatcggacag aggctccgga aggtactgaa
     2581 tctgagatgg aaacccccag tgccattaac gggaatccat catggcacct ggcagatagc
     2641 cccgctgtta atggagccac cggacattcg agctccctgg acgcccgcga ggttatccca
     2701 atggccgcag tcaaacaggc gctgcgtgaa gcaggagatg agtttgaact gcgctatcgg
     2761 cgcgcgttca gtgacctgac ctcacagctg cacattacac ccggcactgc ataccagagt
     2821 tttgagcagg tggttaacga actgttccgc gacggagtga attggggccg tatcgttgcg
     2881 tttttcagtt ttggcggtgc actgtgtgtc gagtcagtgg ataaagaaat gcaggtcctg
     2941 gtgtctcgta ttgcagcttg gatggcaact tatctgaacg atcatctgga gccgtggatt
     3001 caggaaaatg ggggatggga caccttcgtg gaactgtacg gcaacaatgc ggcggctgag
     3061 agtcgtaagg ggcaggagcg gttcaatcgt tggtttctgc agcacaagct gcggaagctg
     3121 aaccctcctg atgagagtgg ccccggctgc atgagctgca agtgtgtgct ctcctgatta
     3181 agcttggtac cgagctcgga tccactagtc cagtgtggtg gaattctgca gatatccagc
     3241 acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     3301 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3361 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3421 taggtgtcat tctattctgg ggggtagggt ggggcaggac agcaaggggg aggattggga
     3481 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3541 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3601 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3661 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3721 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3781 ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag
     3841 tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     3901 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg
     3961 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
     4021 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
     4081 ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc
     4141 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt
     4201 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
     4261 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
     4321 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
     4381 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
     4441 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
     4501 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
     4561 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
     4621 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
     4681 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
     4741 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
     4801 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
     4861 tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta
     4921 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4981 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     5041 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     5101 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     5161 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     5221 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     5281 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5341 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5401 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5461 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5521 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5581 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5641 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5701 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5761 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5821 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5881 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5941 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6001 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     6061 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6121 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6181 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6241 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6301 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6361 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6421 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6481 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6541 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6601 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6661 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6721 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6781 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6841 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6901 aaccaagtca ttctgagaat agtgtatgtg gcgaccgagt tgctcttgcc cggcgtcaat
     6961 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7021 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7081 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     7141 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     7201 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7261 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7321 aaaagtgcca cctgacgtc
//
