LOCUS       sequence-356742-        5802 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutant Ras Binding Domain split-nTEVP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5802)
  AUTHORS   Vlahos AE, Kang J, Aldrete CA, Zhu R, Chong LS, Elowitz MB, Gao XJ
  TITLE     Protease-controlled secretion and display of intercellular signals.
  JOURNAL   Nat Commun. 2022 Feb 17;13(1):912. doi: 10.1038/s41467-022-28623-y.
  PUBMED    35177637
REFERENCE   2  (bases 1 to 5802)
  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 
            Commun."; date: "2022-02-17"; volume: "13(1):912. doi"; pages: " 
            10.1038/s41467-022-28623-y"
COMMENT     Sequence Label: CMVTO-RBDK65E-R89L-nTEVp
FEATURES             Location/Qualifiers
     source          1..5802
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        74..453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        454..657
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     608..628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    659..677
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    680..698
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     708..732
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      824..833
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1474..1503
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1559..1576)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1565..1789
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1961..1980)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2112..2159
                     /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             2167..3189
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2167..3189
                     /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    3319..3440
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3356..3375)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3410..3429
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3489..3505)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3489..3505)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3502..3524)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3513..3529
                     /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(3537..3543,3544..3561,3562..3567))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3582..3603
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3720..3737)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3891..4479)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3971..3990)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4650..5441,5442..5510))
                     /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     5273..5292
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5511..5615)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5690..5709)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgtacgggcc
       61 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      121 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      181 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      241 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      301 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      361 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      421 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      481 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      541 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      601 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
      661 cctatcagtg atagagatct ccctatcagt gatagagatc gtcgacgagc tcgtttagtg
      721 aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg
      781 gaccgatcca gcctccggac tctagcgttt aaacttaagc ttcgccacca tgggatccgg
      841 ggagagtttg tttaaggggc caagggacta taacccaata tcatccacta tttgccacct
      901 cactaacgag agcgatggac atacaacctc tctctacggg ataggcttcg gtcctttcat
      961 cattaccaat aagcatctgt ttcgccgaaa caacggtact cttctggttc aatctcttca
     1021 tggtgtcttc aaggtgaaaa acaccactac gcttcagcaa cacctgattg atggtaggga
     1081 tatgataatt atcagaatgc cgaaagattt cccacctttt ccacagaagc tgaaattcag
     1141 ggaaccgcag agagaggaga ggatttgttt ggtaacgacc aacttccaga cgggctctgg
     1201 cagcagcgcg gatccgagta agacgtcaaa cacaatcagg gtttttctcc ccaatgaaca
     1261 gcggaccgtg gtaaacgtcc gaaacgggat gagtctccac gactgtctga tgaaagcctt
     1321 gaaggtgctg gggcttcagc ctgaatgttg cgctgtcttt cgccttctcc acgaacacaa
     1381 gggcgcaaag gcgaggcttg actggaatac agacgctgca agtttgattg gggaagaact
     1441 tcaggtcgat ttcctctagc tcgaggaggg cccgaacaaa aactcatctc agaagaggat
     1501 ctgaatagcg ccgtcgacca tcatcataat catcattgag tttaaacccg ctgatcagcc
     1561 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     1621 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     1681 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     1741 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatgga gatctatcga
     1801 tgttctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     1861 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1921 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1981 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2041 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2101 tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc
     2161 ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc
     2221 gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc
     2281 gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa
     2341 gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac
     2401 attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg
     2461 ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg
     2521 gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa
     2581 ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg
     2641 tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat
     2701 gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc
     2761 ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag
     2821 gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg
     2881 gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg
     2941 ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt
     3001 gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc
     3061 ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat
     3121 ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca
     3181 aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     3241 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     3301 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3361 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3421 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     3481 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3541 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3601 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3661 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3721 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3781 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3841 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3901 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3961 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4021 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4081 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4141 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4201 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4261 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4321 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4381 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4441 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4501 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4561 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4621 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4681 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4741 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4801 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4861 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4921 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4981 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5041 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5101 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5161 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5221 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5281 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5341 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5401 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5461 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5521 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5581 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5641 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     5701 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     5761 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt ga
//
