LOCUS       sequence-311956-        6301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MESA split-TEV protease chain with FRB Rapamycin ectodomain, CD28 
            transmembrane domain, C-terminal L190K mutant TEV protease, M 
            cleavage sequence, and tTA transcription factor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6301)
  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 6301)
  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-CD28TMD-CTEVp(L190K)-CS(M)-tTA
FEATURES             Location/Qualifiers
     source          1..6301
                     /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"
     CDS             751..816
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1609..1629
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     CDS             join(1648..2268,2269..2652)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
                     EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYG"
     primer_bind     complement(1721..1740)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(2697..2714)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2703..2927
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2973..3401
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3060..3079)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3270..3291
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3424..3620
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3471..3606
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3533..3552
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    3740..3861
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3777..3796)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3831..3850
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3910..3926)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3910..3926)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3923..3945)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3934..3950
                     /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(3958..3964,3965..3982,3983..3988))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4003..4024
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4141..4158)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4312..4900)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4392..4411)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5071..5862,5863..5931))
                     /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     5694..5713
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5932..6036)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6111..6130)
                     /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 tcatgtgccg agccatctct cttaggcgct tgctgctgct gctgctgcag
      721 ctgtcacaac tcctagctgt cactcaaggg gattacaagg atcacgatgg ggattacaaa
      781 gatcatgaca ttgactacaa ggatgacgat gataagatgc tcgagatgtg gcatgaaggc
      841 ctggaagagg catctcgttt gtactttggg gaaaggaacg tgaaaggcat gtttgaggtg
      901 ctggagccct tgcatgctat gatggaacgg ggcccccaga ctctgaagga aacatccttt
      961 aatcaggcct atggtcgaga tttaatggag gcccaagagt ggtgcaggaa gtacatgaaa
     1021 tcagggaatg tcaaggacct cctccaagcc tgggacctct attatcatgt gttccgacga
     1081 atctcaaaga ctagaggagg atctggaggc tctgggggtt caggcggatc aggaggcagt
     1141 ggcgggtcaa ccggtcttgt cgtcgttgca ggggtgctct tctgttatgg tctgttggtg
     1201 acagtcgcac tgtgtgtagg aggcggtagc ggaggcggcg ccaagagcat gtctagcatg
     1261 gtgtcagaca ctagttgcac attcccttca tctgatggca tattctggaa gcattggatt
     1321 caaaccaagg atgggcagtg tggcagtcca ttagtatcaa ctagagatgg gttcattgtt
     1381 ggtatacact cagcatcgaa tttcaccaac acaaacaatt atttcacaag cgtgccgaaa
     1441 aacttcatgg aattgaagac aaatcaggag gcgcagcagt gggttagtgg ttggcgatta
     1501 aatgctgact cagtattgtg ggggggccat aaagttttca tggtgaaacc tgaagagcct
     1561 tttcagccag ttaaggaagc gactcaactc atgaatctta agggctcgga gaacctgtat
     1621 tttcagatgg ggggaggcag cgggggcatg tctagattag ataaaagtaa agtgattaac
     1681 agcgcattag agctgcttaa tgaggtcgga atcgaaggtt taacaacccg taaactcgcc
     1741 cagaagctag gtgtagagca gcctacattg tattggcatg taaaaaataa gcgggctttg
     1801 ctcgacgcct tagccattga gatgttagat aggcaccata ctcacttttg ccctttagaa
     1861 ggggaaagct ggcaagattt tttacgtaat aacgctaaaa gttttagatg tgctttacta
     1921 agtcatcgcg atggagcaaa agtacattta ggtacacggc ctacagaaaa acagtatgaa
     1981 actctcgaaa atcaattagc ctttttatgc caacaaggtt tttcactaga gaatgcatta
     2041 tatgcactca gcgctgtggg gcattttact ttaggttgcg tattggaaga tcaagagcat
     2101 caagtcgcta aagaagaaag ggaaacacct actactgata gtatgccgcc attattacga
     2161 caagctatcg aattatttga tcaccaaggt gcagagccag ccttcttatt cggccttgaa
     2221 ttgatcatat gcggattaga aaaacaactt aaatgtgaaa gtgggtccgc gtacagccgc
     2281 gcgcgtacga aaaacaatta cgggtctacc atcgagggcc tgctcgatct cccggacgac
     2341 gacgcccccg aagaggcggg gctggcggct ccgcgcctgt cctttctccc cgcgggacac
     2401 acgcgcagac tgtcgacggc ccccccgacc gatgtcagcc tgggggacga gctccactta
     2461 gacggcgagg acgtggcgat ggcgcatgcc gacgcgctag acgatttcga tctggacatg
     2521 ttgggggacg gggattcccc gggtccggga tttacccccc acgactccgc cccctacggc
     2581 gctctggata tggccgactt cgagtttgag cagatgttta ccgatgccct tggaattgac
     2641 gagtacggtt aggcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc
     2701 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2761 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2821 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2881 ttgggaagtc aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     2941 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     3001 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3061 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3121 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3181 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3241 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3301 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3361 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3421 cagttaggct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     3481 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     3541 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     3601 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggaggttggg
     3661 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     3721 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     3781 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3841 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     3901 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3961 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4021 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4081 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4141 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4201 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4261 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4321 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4381 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4441 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4501 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4561 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4621 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4681 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4741 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4801 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4861 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4921 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4981 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5041 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5101 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5161 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagatccacg
     5221 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5281 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5341 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5401 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5461 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5521 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5581 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5641 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5701 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5761 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5821 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5881 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5941 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6001 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6061 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     6121 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     6181 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     6241 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     6301 t
//
