LOCUS       sequence-356801-        5973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ER-associated FRB split-cTEVP (Rapalog inducible).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5973)
  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 5973)
  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-p450-FRB-cTEVP
FEATURES             Location/Qualifiers
     source          1..5973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..494
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        495..698
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     649..669
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    700..718
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    721..739
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     749..773
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      865..874
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1686..1715
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1771..1788)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1777..2001
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2173..2192)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2324..2371
                     /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             2379..3401
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2379..3401
                     /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    3531..3652
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3568..3587)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3622..3641
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3701..3717)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3701..3717)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3714..3736)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3725..3741
                     /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(3749..3755,3756..3773,3774..3779))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3794..3815
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3932..3949)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4103..4691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4183..4202)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4862..5653,5654..5722))
                     /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     5485..5504
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5723..5827)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5902..5921)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
       61 tctgcttagg gttaggcgtt ttgcgctgct tcgtacgggc cagatatacg cgttgacatt
      121 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      181 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      241 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      301 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      361 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      421 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      481 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      541 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      601 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      661 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      721 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      781 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      841 ctctagcgtt taaacttaag cttcgccacc atggaccccg ttgtcgtact gggactctgc
      901 ttgtcctgcc tgctcctcct tagtttgtgg aaacaatctt atgggggtgg aaaacttggc
      961 tccgggggag gatccctcga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac
     1021 tttggggaaa ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg
     1081 gaacggggac cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta
     1141 atggaggccc aagagtggtg caggaagtac atgaaatcag ggaatgtcaa ggacctcctc
     1201 caagcctggg acctctatta tcatgtgttc cgacgaatct caaagactag aggaggatct
     1261 ggaggctctg ggggttcagg cggatcagga ggcagtggcg ggtcaaccgg taagagtatg
     1321 agttctatgg tgtccgacac tagctgcacg ttcccctcaa gtgatgggat attctggaaa
     1381 cactggatac agactaaaga cggacagtgt ggaagcccat tggtttccac ccgagatggt
     1441 tttatcgtgg gtatccatag cgcctctaat ttcacaaaca cgaacaacta tttcacttca
     1501 gtgcccaaaa actttatgga gctgctcaca aaccaagaag cgcagcagtg ggtaagcggt
     1561 tggagactta acgctgactc agttctctgg ggggggcaca aagtattcat ggtaaagcca
     1621 gaggagccat tccaaccagt caaagaagcc acacaactta tgaacagcta gctcgaggag
     1681 ggcccgaaca aaaactcatc tcagaagagg atctgaatag cgccgtcgac catcatcata
     1741 atcatcattg agtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     1801 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     1861 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     1921 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     1981 gggatgcggt gggctctatg gagatctatc gatgttctgg ggatgcggtg ggctctatgg
     2041 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     2101 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2161 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2221 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     2281 tcgaccccaa aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa
     2341 gttcctattc tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga
     2401 cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct
     2461 cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc
     2521 gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat
     2581 cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct
     2641 attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc
     2701 ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc
     2761 agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg
     2821 atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca
     2881 ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc
     2941 ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg
     3001 gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg
     3061 tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact
     3121 tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca
     3181 ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg
     3241 cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa
     3301 tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg
     3361 gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg
     3421 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     3481 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     3541 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3601 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     3661 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     3721 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3781 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3841 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3901 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3961 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4021 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4081 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4141 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4201 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4261 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4321 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4381 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4441 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4501 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4561 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4621 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4681 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4741 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4801 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4861 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4921 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4981 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5041 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5101 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5161 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5221 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5281 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5341 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5401 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5461 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5521 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5581 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5641 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5701 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5761 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5821 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     5881 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     5941 gctccctgct tgtgtgttgg aggtcgctga gta
//
