LOCUS       sequence-374570-        7596 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid ensures constitutive expression of the N-terminal fragment 
            of split DHFR (aa 174-186), 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 7596)
  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 7596)
  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: nDHFR173-gs10-NLS-VPR
FEATURES             Location/Qualifiers
     source          1..7596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(70..89)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        261..640
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        641..844
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     795..815
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     889..908
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        889..907
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      970..979
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1531..1551
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1576..1725
                     /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             2194..2550
                     /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             2569..3138
                     /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(3175..3193)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3176..3193)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3213..3230)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3219..3443
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3489..3917
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3576..3595)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3786..3807
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3926..3946)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3931..4260
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4111..4246
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4173..4192
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4327..5121
                     /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(4381..4400)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4991..5010
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5295..5416
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5332..5351)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5386..5405
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5465..5481)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5465..5481)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5478..5500)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5489..5505
                     /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(5513..5519,5520..5537,5538..5543))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5558..5579
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5696..5713)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5867..6455)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5947..5966)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6626..7417,7418..7486))
                     /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     7249..7268
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7487..7591)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
       61 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
      121 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
      181 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
      241 acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac
      301 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      361 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      421 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      481 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      541 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      601 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      661 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
      721 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
      781 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
      841 agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca
      901 ctatagggag acccaagctt ggtaccgagc tcggatccac tagtaacggc cgccagtgtg
      961 ctggaattcg ccaccatggt aggaagcctc aattgcatag tcgctgtgag tcaaaatatg
     1021 ggaattggca aaaatggcga tctcccttgg cccccactga gaaacgaatt taggtatttt
     1081 caacggatga ccactacttc tagcgtggaa gggaaacaga acctcgtgat aatgggtaaa
     1141 aagacttggt ttagcattcc ggaaaaaaat cggccactta agggacgaat aaatcttgtc
     1201 ttgtcaaggg aattgaagga gccgccgcag ggtgcccact ttctcagtcg gtcactggac
     1261 gatgcgctga agctgaccga acagcccgag ctcgccaaca aggtggatat ggtgtggata
     1321 gtcggcggta gttcagtcta caaagaagct atgaaccatc cggggcattt gaaattgttt
     1381 gttacgcgca ttatgcaaga ttttgaaagt gatacctttt tcccggagat agaccttgaa
     1441 aaatataagt tgcttcctga gtatccggga gtgctctcag atgtgcaaga agagaagggc
     1501 ggctctggcg gcggctccgg aggctctgat ccaaaaaaga agagaaaggt agaggccagc
     1561 ggttcgggac gggctgacgc attggacgat tttgatctgg atatgctggg aagtgacgcc
     1621 ctcgatgatt ttgaccttga catgcttggt tcggatgccc ttgatgactt tgacctcgac
     1681 atgctcggca gtgacgccct tgatgatttc gacctggaca tgctgattaa cactagtggc
     1741 ggctctggcg gcggctccgg aggctctagc cagtacctgc ccgacaccga cgaccggcac
     1801 cggatcgagg aaaagcggaa gcggacctac gagacattca agagcatcat gaagaagtcc
     1861 cccttcagcg gccccaccga ccctagacct ccacctagaa gaatcgccgt gcccagcaga
     1921 tccagcgcca gcgtgccaaa acctgccccc cagccttacc ccttcaccag cagcctgagc
     1981 accatcaact acgacgagtt ccctaccatg gtgttcccca gcggccagat ctctcaggcc
     2041 tctgctctgg ctccagcccc tcctcaggtg ctgcctcagg ctcctgctcc tgcaccagct
     2101 ccagccatgg tgtctgcact ggctcaggca ccagcacccg tgcctgtgct ggctcctgga
     2161 cctccacagg ctgtggctcc accagcccct aaacctacac aggccggcga gggcacactg
     2221 tctgaagctc tgctgcagct gcagttcgac gacgaggatc tgggagccct gctgggaaac
     2281 agcaccgatc ctgccgtgtt caccgacctg gccagcgtgg acaacagcga gttccagcag
     2341 ctgctgaacc agggcatccc tgtggcccct cacaccaccg agcccatgct gatggaatac
     2401 cccgaggcca tcacccggct cgtgacaggc gctcagaggc ctcctgatcc agctcctgcc
     2461 cctctgggag caccaggcct gcctaatgga ctgctgtctg gcgacgagga cttcagctct
     2521 atcgccgata tggatttctc agccttgctg ggctctggca gcggcagccg ggattccagg
     2581 gaagggatgt ttttgccgaa gcctgaggcc ggctccgcta ttagtgacgt gtttgagggc
     2641 cgcgaggtgt gccagccaaa acgaatccgg ccatttcatc ctccaggaag tccatgggcc
     2701 aaccgcccac tccccgccag cctcgcacca acaccaaccg gtccagtaca tgagccagtc
     2761 gggtcactga ccccggcacc agtccctcag ccactggacc cagcgcccgc agtgactccc
     2821 gaggccagtc acctgttgga ggaccccgat gaagagacga gccaggctgt caaagccctt
     2881 cgggagatgg ccgatactgt gattccccag aaggaagagg ctgcaatctg tggccaaatg
     2941 gacctttccc atccgccccc aaggggccat ctggatgagc tgacaaccac acttgagtcc
     3001 atgaccgagg atctgaacct ggactcaccc ctgaccccgg aattgaacga gattctggat
     3061 accttcctga acgacgagtg cctcttgcat gccatgcata tcagcacagg actgtccatc
     3121 ttcgacacat ctctgtttga cacatacagg tatatctaat ctagagggcc ctattctata
     3181 gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     3241 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3301 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3361 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3421 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3481 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3541 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3601 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3661 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3721 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3781 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3841 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3901 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3961 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     4021 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4081 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4141 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     4201 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4261 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     4321 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4381 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4441 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4501 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4561 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4621 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4681 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4741 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4801 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     4861 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4921 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4981 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     5041 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     5101 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     5161 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     5221 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5281 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5341 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5401 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     5461 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5521 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5581 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5641 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5701 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5761 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5821 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5881 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5941 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     6001 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6061 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6121 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6181 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6241 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6301 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6361 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6421 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6481 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6541 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6601 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6661 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6721 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6781 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6841 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6901 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6961 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     7021 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     7081 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7141 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7201 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7261 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7321 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7381 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7441 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7501 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7561 tttagaaaaa taaacaaata ggggttccgc gcacat
//
