LOCUS       sequence-374529-        7716 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid ensures constitutive expression of the N-terminal fragment 
            of split TRbeta (aa 200-412), fused to the VPR activation domain, a 
            flexible GS linker and the SV40 NLS signal at the C-terminus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7716)
  AUTHORS   Rihtar E, Lebar T, Lainscek D, Kores K, Lesnik S, Bren U, Jerala R
  TITLE     Chemically inducible split protein regulators for mammalian cells.
  JOURNAL   Nat Chem Biol. 2022 Sep 26. doi: 10.1038/s41589-022-01136-x.
  PUBMED    36163385
REFERENCE   2  (bases 1 to 7716)
  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 Chem 
            Biol. 2022 Sep 26. doi: 10.1038/s41589-022-01136-x."
COMMENT     Sequence Label: nTRb412-gs10-NLS-VPR
FEATURES             Location/Qualifiers
     source          1..7716
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(80..99)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        271..650
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        651..854
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     805..825
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     899..918
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        899..917
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1661..1681
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1706..1855
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             2324..2680
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             2699..3268
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     promoter        complement(3305..3323)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3306..3323)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3343..3360)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3349..3573
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3619..4047
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3706..3725)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3916..3937
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4056..4076)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4061..4390
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4241..4376
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4303..4322
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4457..5251
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4511..4530)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5121..5140
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5425..5546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5462..5481)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5516..5535
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5595..5611)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5595..5611)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5608..5630)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5619..5635
                     /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(5643..5649,5650..5667,5668..5673))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5688..5709
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5826..5843)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5997..6585)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6077..6096)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6756..7547,7548..7616))
                     /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     7379..7398
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7617..7716,1..5))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
       61 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
      121 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
      181 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
      241 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
      301 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      361 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      421 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      481 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      541 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      601 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      661 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      721 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      781 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      841 atataagcag agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta
      901 atacgactca ctatagggag acccaagctt ggtaccgagc tcggatccac tagtaacggc
      961 cgccagtgtg ctggaattcg ccaccatgca catggaggag ctgcagaaga gcatcggcca
     1021 caagcccgag cccaccgacg aggagtggga gctgatcaag accgtgaccg aggcccacgt
     1081 ggccaccaac gcccagggca gccactggaa gcagaagagg aagttcctgc ccgaggacat
     1141 cggccaggcc cccatcgtga acgcccccga gggcggcaag gtggacctgg aggccttcag
     1201 ccacttcacc aagatcatca cccccgccat caccagggtg gtggacttcg ccaagaagct
     1261 gcccatgttc tgcgagctgc cctgcgagga ccagatcatc ctgctgaagg gctgctgcat
     1321 ggagatcatg agcctgaggg ccgccgtgag gtacgacccc gagagcgaga ccctgaccct
     1381 gaacggcgag atggccgtga ccaggggcca gctgaagaac ggcggcctgg gcgtggtgag
     1441 cgacgccatc ttcgacctgg gcatgagcct gagcagcttc aacctggacg acaccgaggt
     1501 ggccctgctg caggccgtgc tgctgatgag cagcgacagg cccggcctgg cctgcgtgga
     1561 gaggatcgag aagtaccagg acagcttcct gctggccttc gagcactaca tcaactacag
     1621 gaagcacggc ggctctggcg gcggctccgg aggctctgat ccaaaaaaga agagaaaggt
     1681 agaggccagc ggttcgggac gggctgacgc attggacgat tttgatctgg atatgctggg
     1741 aagtgacgcc ctcgatgatt ttgaccttga catgcttggt tcggatgccc ttgatgactt
     1801 tgacctcgac atgctcggca gtgacgccct tgatgatttc gacctggaca tgctgattaa
     1861 cactagtggc ggctctggcg gcggctccgg aggctctagc cagtacctgc ccgacaccga
     1921 cgaccggcac cggatcgagg aaaagcggaa gcggacctac gagacattca agagcatcat
     1981 gaagaagtcc cccttcagcg gccccaccga ccctagacct ccacctagaa gaatcgccgt
     2041 gcccagcaga tccagcgcca gcgtgccaaa acctgccccc cagccttacc ccttcaccag
     2101 cagcctgagc accatcaact acgacgagtt ccctaccatg gtgttcccca gcggccagat
     2161 ctctcaggcc tctgctctgg ctccagcccc tcctcaggtg ctgcctcagg ctcctgctcc
     2221 tgcaccagct ccagccatgg tgtctgcact ggctcaggca ccagcacccg tgcctgtgct
     2281 ggctcctgga cctccacagg ctgtggctcc accagcccct aaacctacac aggccggcga
     2341 gggcacactg tctgaagctc tgctgcagct gcagttcgac gacgaggatc tgggagccct
     2401 gctgggaaac agcaccgatc ctgccgtgtt caccgacctg gccagcgtgg acaacagcga
     2461 gttccagcag ctgctgaacc agggcatccc tgtggcccct cacaccaccg agcccatgct
     2521 gatggaatac cccgaggcca tcacccggct cgtgacaggc gctcagaggc ctcctgatcc
     2581 agctcctgcc cctctgggag caccaggcct gcctaatgga ctgctgtctg gcgacgagga
     2641 cttcagctct atcgccgata tggatttctc agccttgctg ggctctggca gcggcagccg
     2701 ggattccagg gaagggatgt ttttgccgaa gcctgaggcc ggctccgcta ttagtgacgt
     2761 gtttgagggc cgcgaggtgt gccagccaaa acgaatccgg ccatttcatc ctccaggaag
     2821 tccatgggcc aaccgcccac tccccgccag cctcgcacca acaccaaccg gtccagtaca
     2881 tgagccagtc gggtcactga ccccggcacc agtccctcag ccactggacc cagcgcccgc
     2941 agtgactccc gaggccagtc acctgttgga ggaccccgat gaagagacga gccaggctgt
     3001 caaagccctt cgggagatgg ccgatactgt gattccccag aaggaagagg ctgcaatctg
     3061 tggccaaatg gacctttccc atccgccccc aaggggccat ctggatgagc tgacaaccac
     3121 acttgagtcc atgaccgagg atctgaacct ggactcaccc ctgaccccgg aattgaacga
     3181 gattctggat accttcctga acgacgagtg cctcttgcat gccatgcata tcagcacagg
     3241 actgtccatc ttcgacacat ctctgtttga cacatacagg tatatctaat ctagagggcc
     3301 ctattctata gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta
     3361 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3421 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3481 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3541 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     3601 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3661 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3721 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3781 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3841 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccttttg acgttggagt
     3901 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3961 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     4021 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     4081 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4141 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4201 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     4261 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4321 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4381 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     4441 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4501 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4561 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4621 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4681 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4741 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4801 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4861 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4921 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4981 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     5041 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     5101 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     5161 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     5221 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     5281 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     5341 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     5401 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     5461 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     5521 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     5581 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     5641 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     5701 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5761 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5821 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5881 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5941 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     6001 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     6061 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     6121 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     6181 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     6241 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     6301 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     6361 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6421 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6481 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6541 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6601 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6661 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6721 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6781 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6841 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6901 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6961 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     7021 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     7081 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     7141 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     7201 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     7261 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     7321 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     7381 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7441 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7501 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7561 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7621 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     7681 tttgaatgta tttagaaaaa taaacaaata ggggtt
//
