LOCUS       sequence-311962-        5134 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cytosolic split-TEV protease chain with FRB Rapamycin sensing 
            domain, C-terminal WT TEV protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5134)
  AUTHORS   Dolberg TB, Meger AT, Boucher JD, Corcoran WK, Schauer EE, Prybutok 
            AN, Raman S, Leonard JN
  TITLE     Computation-guided optimization of split protein systems.
  JOURNAL   Nat Chem Biol. 2021 May;17(5):531-539. doi: 
            10.1038/s41589-020-00729-8. Epub 2021 Feb 1.
  PUBMED    33526893
REFERENCE   2  (bases 1 to 5134)
  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/s41589-020-00729-8"; journalName: "Nat Chem Biol"; date: 
            "2021-05"; volume: "17"; issue: "5"; pages: "531-539"
COMMENT     Sequence Label: FRB-CTEVp(WT)
FEATURES             Location/Qualifiers
     source          1..5134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      667..676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             676..741
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1530..1547)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1536..1760
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1806..2234
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1893..1912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2103..2124
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2257..2453
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2304..2439
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2366..2385
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2573..2694
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2610..2629)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2664..2683
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2743..2759)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2743..2759)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2756..2778)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2767..2783
                     /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(2791..2797,2798..2815,2816..2821))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2836..2857
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2974..2991)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3145..3733)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3225..3244)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3904..4695,4696..4764))
                     /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     4527..4546
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4765..4869)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4944..4963)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag tcccaagctg
      661 gctagcgcca ccatggacta caaagaccac gacggcgatt acaaggatca tgacatcgat
      721 tacaaagacg atgacgataa gatggaagag ctgagccagg ctctggccag ctcctttagc
      781 gtgtcctccg gaatgcttga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac
      841 tttggggaaa ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg
      901 gaacggggac cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta
      961 atggaggccc aagagtggtg caggaagtac atgaaatcag ggaatgtcaa ggacctcctc
     1021 caagcctggg acctctatta tcatgtgttc cgacgaatct caaagactag aggatccggt
     1081 ggtggcgggt ctggaggagg aggtagtggt ggaggtggct ctggtaccaa gagcatgtct
     1141 agcatggtgt cagacactag ttgcacattc ccttcatctg atggcatatt ctggaagcat
     1201 tggattcaaa ccaaggatgg gcagtgtggc agtccattag tatcaactag agatgggttc
     1261 attgttggta tacactcagc atcgaatttc accaacacaa acaattattt cacaagcgtg
     1321 ccgaaaaact tcatggaatt gttgacaaat caggaggcgc agcagtgggt tagtggttgg
     1381 cgattaaatg ctgactcagt attgtggggg ggccataaag ttttcatggt gaaacctgaa
     1441 gagccttttc agccagttaa ggaagcgact caactcatga attaagcggc cgctcgagtc
     1501 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     1561 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1621 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     1681 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gtcaatagca ggcatgctgg
     1741 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     1801 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1861 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1921 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1981 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2041 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2101 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2161 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2221 atttaacgcg aattaattct gtggaatgtg tgtcagttag gctcaattag tcagcaacca
     2281 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     2341 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     2401 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     2461 cgggagcttg tatatccatt ttcggaggtt gggcttcgga atcgttttcc gggacgccgg
     2521 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     2581 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2641 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     2701 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     2761 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     2821 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     2881 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     2941 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3001 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3061 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3121 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3181 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3241 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3301 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3361 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3421 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3481 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3541 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3601 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3661 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3721 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3781 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     3841 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3901 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3961 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4021 ccccagtgct gcaatgatac cgcgagatcc acgctcaccg gctccagatt tatcagcaat
     4081 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4141 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4201 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4261 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4321 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4381 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4441 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4501 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4561 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4621 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4681 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4741 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     4801 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4861 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc
     4921 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     4981 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
     5041 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
     5101 ctgcttcgcg atgtacgggc cagatatacg cgtt
//
