LOCUS       sequence-269546-        7593 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the PYL1 abscisic acid-inducible domain fused to the VPR 
            transcriptional activation domain in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7593)
  AUTHORS   Lebar T, Lainscek D, Merljak E, Aupic J, Jerala R
  TITLE     A tunable orthogonal coiled-coil interaction toolbox for engineering
            mammalian cells.
  JOURNAL   Nat Chem Biol. 2020 Jan 6. pii: 10.1038/s41589-019-0443-y. doi: 
            10.1038/s41589-019-0443-y.
  PUBMED    31907374
REFERENCE   2  (bases 1 to 7593)
  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. 2020 Jan 6. pii: 10.1038/s41589-019-0443-y. doi: 
            10.1038/s41589-019-0443-y."
COMMENT     Sequence Label: pcDNA3-His:PYL1:nls:VPR
FEATURES             Location/Qualifiers
     source          1..7593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             953..970
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1520..1540
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1565..1714
                     /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             2183..2539
                     /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             2558..3127
                     /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(3146..3164)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3147..3164)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3184..3201)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3190..3414
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3460..3888
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3547..3566)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3757..3778
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3897..3917)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3902..4231
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4082..4217
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4144..4163
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4298..5092
                     /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(4352..4371)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4962..4981
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5266..5387
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5303..5322)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5357..5376
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5436..5452)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5436..5452)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5449..5471)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5460..5476
                     /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(5484..5490,5491..5508,5509..5514))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5529..5550
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5667..5684)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5838..6426)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5918..5937)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6597..7388,7389..7457))
                     /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     7220..7239
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7458..7562)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 tagctcggat ccactagtaa cggccgccag tgtgctggaa ttcgccacca tgcaccacca
      961 ccaccaccac ggtgggggcg cgccaactca agacgaattc acccaactct cccaatcaat
     1021 cgccgagttc cacacgtacc aactcggtaa cggccgttgc tcatctctcc tagctcagcg
     1081 aatccacgcg ccgccggaaa cagtatggtc cgtggtgaga cgtttcgata ggccacagat
     1141 ttacaaacac ttcatcaaaa gctgtaacgt gagtgaagat ttcgagatgc gagtgggatg
     1201 cacgcgcgac gtgaacgtga taagtggatt accggcgaat acgtctcgag agagattaga
     1261 tctgttggac gatgatcgga gagtgactgg gtttagtata accggtggtg aacataggct
     1321 gaggaattat aaatcggtta cgacggttca tagatttgag aaagaagaag aagaagaaag
     1381 gatctggacc gttgttttgg aatcttatgt tgttgatgta ccggaaggta attcggagga
     1441 agatacgaga ttgtttgctg atacggttat tagattgaat cttcagaaac ttgcttcgat
     1501 cactgaagct atgaacgatc caaaaaagaa gagaaaggta gaggccagcg gttcgggacg
     1561 ggctgacgca ttggacgatt ttgatctgga tatgctggga agtgacgccc tcgatgattt
     1621 tgaccttgac atgcttggtt cggatgccct tgatgacttt gacctcgaca tgctcggcag
     1681 tgacgccctt gatgatttcg acctggacat gctgattaac actagtggcg gctctggcgg
     1741 cggctccgga ggctctagcc agtacctgcc cgacaccgac gaccggcacc ggatcgagga
     1801 aaagcggaag cggacctacg agacattcaa gagcatcatg aagaagtccc ccttcagcgg
     1861 ccccaccgac cctagacctc cacctagaag aatcgccgtg cccagcagat ccagcgccag
     1921 cgtgccaaaa cctgcccccc agccttaccc cttcaccagc agcctgagca ccatcaacta
     1981 cgacgagttc cctaccatgg tgttccccag cggccagatc tctcaggcct ctgctctggc
     2041 tccagcccct cctcaggtgc tgcctcaggc tcctgctcct gcaccagctc cagccatggt
     2101 gtctgcactg gctcaggcac cagcacccgt gcctgtgctg gctcctggac ctccacaggc
     2161 tgtggctcca ccagccccta aacctacaca ggccggcgag ggcacactgt ctgaagctct
     2221 gctgcagctg cagttcgacg acgaggatct gggagccctg ctgggaaaca gcaccgatcc
     2281 tgccgtgttc accgacctgg ccagcgtgga caacagcgag ttccagcagc tgctgaacca
     2341 gggcatccct gtggcccctc acaccaccga gcccatgctg atggaatacc ccgaggccat
     2401 cacccggctc gtgacaggcg ctcagaggcc tcctgatcca gctcctgccc ctctgggagc
     2461 accaggcctg cctaatggac tgctgtctgg cgacgaggac ttcagctcta tcgccgatat
     2521 ggatttctca gccttgctgg gctctggcag cggcagccgg gattccaggg aagggatgtt
     2581 tttgccgaag cctgaggccg gctccgctat tagtgacgtg tttgagggcc gcgaggtgtg
     2641 ccagccaaaa cgaatccggc catttcatcc tccaggaagt ccatgggcca accgcccact
     2701 ccccgccagc ctcgcaccaa caccaaccgg tccagtacat gagccagtcg ggtcactgac
     2761 cccggcacca gtccctcagc cactggaccc agcgcccgca gtgactcccg aggccagtca
     2821 cctgttggag gaccccgatg aagagacgag ccaggctgtc aaagcccttc gggagatggc
     2881 cgatactgtg attccccaga aggaagaggc tgcaatctgt ggccaaatgg acctttccca
     2941 tccgccccca aggggccatc tggatgagct gacaaccaca cttgagtcca tgaccgagga
     3001 tctgaacctg gactcacccc tgaccccgga attgaacgag attctggata ccttcctgaa
     3061 cgacgagtgc ctcttgcatg ccatgcatat cagcacagga ctgtccatct tcgacacatc
     3121 tctgttttga tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat
     3181 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3241 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     3301 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3361 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     3421 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3481 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3541 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3601 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3661 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3721 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3781 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3841 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     3901 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3961 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     4021 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     4081 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4141 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     4201 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     4261 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     4321 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     4381 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     4441 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     4501 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     4561 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     4621 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     4681 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     4741 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     4801 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     4861 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     4921 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4981 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     5041 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     5101 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     5161 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     5221 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     5281 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     5341 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     5401 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     5461 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     5521 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5581 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5641 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5701 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5761 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5821 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5881 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5941 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     6001 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6061 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6121 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6181 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6241 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     6301 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6361 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6421 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6481 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6541 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6601 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6661 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6721 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6781 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6841 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6901 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6961 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     7021 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     7081 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7141 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7201 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7261 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7321 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     7381 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     7441 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     7501 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7561 cgcacatttc cccgaaaagt gccacctgac gtc
//
