LOCUS       sequence-374534-        7601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid ensures constitutive expression of the N-terminal fragment 
            of split GR2 (aa 1-175), 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 7601)
  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 7601)
  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: nGR2175-gs10-NLS-VPR
FEATURES             Location/Qualifiers
     source          1..7601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(50..69)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        241..620
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        621..824
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     775..795
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     869..888
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        869..887
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1517..1537
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1562..1711
                     /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             2180..2536
                     /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             2555..3124
                     /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(3161..3179)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3162..3179)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3199..3216)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3205..3429
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3475..3903
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3562..3581)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3772..3793
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3912..3932)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3917..4245
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4097..4231
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4158..4177
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4312..5106
                     /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(4366..4385)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4976..4995
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5280..5401
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5317..5336)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5371..5390
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5450..5466)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5450..5466)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5463..5485)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5474..5490
                     /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(5498..5504,5505..5522,5523..5528))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5543..5564
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5681..5698)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5852..6440)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5932..5951)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6611..7402,7403..7471))
                     /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     7234..7253
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7472..7576)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
       61 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
      121 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
      181 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
      241 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      301 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      361 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      421 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      481 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      541 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      601 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      661 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      721 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      781 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      841 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      901 ggtaccgagc tcggatccac tagtaacggc cgccagtgtg ctggaattcg ccaccatgct
      961 gatcagcctg ctggaggtga tcgagcccga ggtgctgtac agcggctacg acagcaccct
     1021 gcccgacacc agcaccaggc tgatgagcac cctgaacagg ctgggcggca ggcaggtggt
     1081 gagcgccgtg aagtgggcca aggccctgcc cggcttcagg aacctgcacc tggacgacca
     1141 gatgaccctg ctgcagtaca gctggatgag cctgatggcc ttcagcctgg gctggaggag
     1201 ctacaagcag agcaacggca acatgctgtg cttcgccccc gacctggtga tcaacgagga
     1261 gaggatgcag ctgccctaca tgtacgacca gtgccagcag atgctgaaga tcagcagcga
     1321 gttcgtgagg ctgcaggtga gctacgacga gtacctgtgc atgaaggtgc tgctgctgct
     1381 gagcaccgtg cccaaggacg gcctgaagag ccaggccgtg ttcgacgaga tcaggatgac
     1441 ctacatcaag gagctgggca aggccatcgt gaagagggag ggcggcggct ctggcggcgg
     1501 ctccggaggc tctgatccaa aaaagaagag aaaggtagag gccagcggtt cgggacgggc
     1561 tgacgcattg gacgattttg atctggatat gctgggaagt gacgccctcg atgattttga
     1621 ccttgacatg cttggttcgg atgcccttga tgactttgac ctcgacatgc tcggcagtga
     1681 cgcccttgat gatttcgacc tggacatgct gattaacact agtggcggct ctggcggcgg
     1741 ctccggaggc tctagccagt acctgcccga caccgacgac cggcaccgga tcgaggaaaa
     1801 gcggaagcgg acctacgaga cattcaagag catcatgaag aagtccccct tcagcggccc
     1861 caccgaccct agacctccac ctagaagaat cgccgtgccc agcagatcca gcgccagcgt
     1921 gccaaaacct gccccccagc cttacccctt caccagcagc ctgagcacca tcaactacga
     1981 cgagttccct accatggtgt tccccagcgg ccagatctct caggcctctg ctctggctcc
     2041 agcccctcct caggtgctgc ctcaggctcc tgctcctgca ccagctccag ccatggtgtc
     2101 tgcactggct caggcaccag cacccgtgcc tgtgctggct cctggacctc cacaggctgt
     2161 ggctccacca gcccctaaac ctacacaggc cggcgagggc acactgtctg aagctctgct
     2221 gcagctgcag ttcgacgacg aggatctggg agccctgctg ggaaacagca ccgatcctgc
     2281 cgtgttcacc gacctggcca gcgtggacaa cagcgagttc cagcagctgc tgaaccaggg
     2341 catccctgtg gcccctcaca ccaccgagcc catgctgatg gaataccccg aggccatcac
     2401 ccggctcgtg acaggcgctc agaggcctcc tgatccagct cctgcccctc tgggagcacc
     2461 aggcctgcct aatggactgc tgtctggcga cgaggacttc agctctatcg ccgatatgga
     2521 tttctcagcc ttgctgggct ctggcagcgg cagccgggat tccagggaag ggatgttttt
     2581 gccgaagcct gaggccggct ccgctattag tgacgtgttt gagggccgcg aggtgtgcca
     2641 gccaaaacga atccggccat ttcatcctcc aggaagtcca tgggccaacc gcccactccc
     2701 cgccagcctc gcaccaacac caaccggtcc agtacatgag ccagtcgggt cactgacccc
     2761 ggcaccagtc cctcagccac tggacccagc gcccgcagtg actcccgagg ccagtcacct
     2821 gttggaggac cccgatgaag agacgagcca ggctgtcaaa gcccttcggg agatggccga
     2881 tactgtgatt ccccagaagg aagaggctgc aatctgtggc caaatggacc tttcccatcc
     2941 gcccccaagg ggccatctgg atgagctgac aaccacactt gagtccatga ccgaggatct
     3001 gaacctggac tcacccctga ccccggaatt gaacgagatt ctggatacct tcctgaacga
     3061 cgagtgcctc ttgcatgcca tgcatatcag cacaggactg tccatcttcg acacatctct
     3121 gtttgacaca tacaggtata tctaatctag agggccctat tctatagtgt cacctaaatg
     3181 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     3241 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     3301 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     3361 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     3421 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     3481 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3541 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3601 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3661 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3721 cctgatagac ggtttttcgc cttttgacgt tggagtccac gttctttaat agtggactct
     3781 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3841 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3901 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3961 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     4021 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     4081 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     4141 tggctgacta atttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     4201 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     4261 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     4321 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4381 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4441 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     4501 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4561 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4621 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4681 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4741 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4801 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     4861 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4921 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4981 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     5041 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     5101 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     5161 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     5221 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     5281 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     5341 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     5401 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     5461 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5521 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5581 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5641 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5701 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5761 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5821 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5881 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5941 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6001 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6061 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6121 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6181 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6241 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6301 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6361 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6421 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6481 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6541 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6601 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6661 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6721 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6781 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6841 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6901 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6961 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7021 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7081 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7141 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7201 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7261 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7321 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7381 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7441 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7501 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7561 aaataggggt tccgcgcaca tttccccgaa aagtgccacc t
//
