LOCUS       sequence-311953-        5416 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MESA split-TEV protease chain with FKBP Rapamycin ectodomain, CD28 
            transmembrane domain, N-terminal H75E mutant TEV protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5416)
  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 5416)
  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: FKBP-CD28TMD-NTEVp(H75E)
FEATURES             Location/Qualifiers
     source          1..5416
                     /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             join(817..819,820..1140)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     misc_feature    1202..1254
                     /label=MCS
                     /note="multiple cloning site"
     primer_bind     complement(1812..1829)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1818..2042
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2088..2516
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2175..2194)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2385..2406
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2539..2735
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2586..2721
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2648..2667
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2855..2976
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2892..2911)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2946..2965
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3025..3041)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3025..3041)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3038..3060)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3049..3065
                     /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(3073..3079,3080..3097,3098..3103))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3118..3139
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3256..3273)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3427..4015)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3507..3526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4186..4977,4978..5046))
                     /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     4809..4828
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5047..5151)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5226..5245)
                     /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 gataagatgg gagtgcaggt ggaaaccatc
      841 tccccaggag acgggcgcac cttccccaag cgcggccaga cctgcgtggt gcactacacc
      901 gggatgcttg aagatggaaa gaaatttgat tcctcccggg acagaaacaa gccctttaag
      961 tttatgctag gcaagcagga ggtgatccga ggctgggaag aaggggttgc ccagatgagt
     1021 gtgggtcaga gagccaaact gactatatct ccagattatg cctatggtgc cactgggcac
     1081 ccaggcatca tcccaccaca tgccactctc gtcttcgatg tggagcttct aaaactggaa
     1141 agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggt
     1201 cctcgagctc aagcttcgaa ttctgcagtc gacggtaccg cgggcccggg atccaccgga
     1261 tctagaggcg gatctggagg ctctgggggt tcaggcggat caggaggcag tggcgggtca
     1321 accggtcttg tcgtcgttgc aggggtgctc ttctgttatg gtctgttggt gacagtcgca
     1381 ctgtgtgtag gaggcggtag cggaggcggc gccgagagct tgtttaaggg gccgcgtgat
     1441 tacaacccga tatcgagcac catttgtcat ttgacgaatg aatctgatgg gcacacaaca
     1501 tcgttgtatg gtattggatt tggtcccttc atcattacaa acaagcactt gtttagaaga
     1561 aataatggaa cactgttggt ccaatcacta catggtgtat tcaaggtcaa gaacaccacg
     1621 actttgcaac aagagctcat tgatgggagg gacatgataa ttattcgcat gcctaaggat
     1681 ttcccaccat ttcctcaaaa gctgaaattt agagagccac aaagggaaga gcgcatatgt
     1741 cttgtgacaa ccaacttcca aacttaggcg gccgctcgag tctagagggc ccgtttaaac
     1801 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     1861 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     1921 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     1981 cagcaagggg gaggattggg aagtcaatag caggcatgct ggggatgcgg tgggctctat
     2041 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     2101 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2161 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2221 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2281 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2341 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2401 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2461 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     2521 ctgtggaatg tgtgtcagtt aggctcaatt agtcagcaac catagtcccg cccctaactc
     2581 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     2641 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     2701 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     2761 ttttcggagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     2821 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     2881 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     2941 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     3001 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     3061 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     3121 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3181 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3241 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3301 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3361 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3421 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3481 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3541 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3601 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3661 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3721 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3781 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3841 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3901 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3961 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4021 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4081 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4141 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4201 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4261 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4321 accgcgagat ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4381 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4441 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4501 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4561 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4621 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4681 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4741 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4801 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4861 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4921 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4981 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5041 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5101 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5161 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
     5221 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
     5281 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
     5341 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
     5401 gccagatata cgcgtt
//
