LOCUS       sequence-322045-        8864 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TU for the constitutive expression of a chimeric construct 
            comprising a DNA binding protein fused to A. thaliana PIF6 (amino 
            acids 1-100) and a nuclear localization sequence. Part of a red 
            light-controlled synthetic genetic switch.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8864)
  AUTHORS   Calvache C, Vazquez-Vilar M, Selma S, Uranga M, Fernandez-Del-Carmen
            A, Daros JA, Orzaez D
  TITLE     Strong and tunable anti-CRISPR/Cas activities in plants.
  JOURNAL   Plant Biotechnol J. 2022 Feb;20(2):399-408. doi: 10.1111/pbi.13723. 
            Epub 2021 Oct 31.
  PUBMED    34632687
REFERENCE   2  (bases 1 to 8864)
  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: "10.1111/pbi.13723";
            journalName: "Plant Biotechnol J"; date: "2022-02"; volume: "20"; 
            issue: "2"; pages: "399-408"
COMMENT     Sequence Label: pEGB3 alpha1 35s:PIF6:tNos (GB0170)
FEATURES             Location/Qualifiers
     source          1..8864
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             598..1671
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      1737..1931
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(2167..2189)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2275..2415
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2330..2349
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2430..2447)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2601..3189)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2681..2700)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3277..4092)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3277..4092)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4000..4019
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    4525..4549
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        5182..5526
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     5470..5490
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             6539..6559
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      6586..6838
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(6873..6891)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    7079..7103
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             join(8404..8864,1..169)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
ORIGIN
        1 ggcgctggcc gggtacgagc tgcccattct tgagtcccgt atcacgcagc gcgtgagcta
       61 cccaggcact gccgccgccg gcacaaccgt tcttgaatca gaacccgagg gcgacgctgc
      121 ccgcgaggtc caggcgctgg ccgctgaaat taaatcaaaa ctcatttgag ttaatgaggt
      181 aaagagaaaa tgagcaaaag cacaaacacg ctaagtgccg gccgtccgag cgcacgcagc
      241 agcaaggctg caacgttggc cagcctggca gacacgccag ccatgaagcg ggtcaacttt
      301 cagttgccgg cggaggatca caccaagctg aagatgtacg cggtacgcca aggcaagacc
      361 attaccgagc tgctatctga atacatcgcg cagctaccag agtaaatgag caaatgaata
      421 aatgagtaga tgaattttag cggctaaagg aggcggcatg gaaaatcaag aacaaccagg
      481 caccgacgcc gtggaatgcc ccatgtgtgg aggaacgggc ggttggccag gcgtaagcgg
      541 ctgggttgcc tgccggccct gcaatggcac tggaaccccc aagcccgagg aatcggcgtg
      601 agcggtcgca aaccatccgg cccggtacaa atcggcgcgg cgctgggtga tgacctggtg
      661 gagaagttga aggccgcgca ggccgcccag cggcaacgca tcgaggcaga agcacgcccc
      721 ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag aatcccggca accgccggca
      781 gccggtgcgc cgtcgattag gaagccgccc aagggcgacg agcaaccaga ttttttcgtt
      841 ccgatgctct atgacgtggg cacccgcgat agtcgcagca tcatggacgt ggccgttttc
      901 cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc gctacgagct tccagacggg
      961 cacgtagagg tttccgcagg gccggccggc atggccagtg tgtgggatta cgacctggta
     1021 ctgatggcgg tttcccatct aaccgaatcc atgaaccgat accgggaagg gaagggagac
     1081 aagcccggcc gcgtgttccg tccacacgtt gcggacgtac tcaagttctg ccggcgagcc
     1141 gatggcggaa agcagaaaga cgacctggta gaaacctgca ttcggttaaa caccacgcac
     1201 gttgccatgc agcgtacgaa gaaggccaag aacggccgcc tggtgacggt atccgagggt
     1261 gaagccttga ttagccgcta caagatcgta aagagcgaaa ccgggcggcc ggagtacatc
     1321 gagatcgagc tagctgattg gatgtaccgc gagatcacag aaggcaagaa cccggacgtg
     1381 ctgacggttc accccgatta ctttttgatc gatcccggca tcggccgttt tctctaccgc
     1441 ctggcacgcc gcgccgcagg caaggcagaa gccagatggt tgttcaagac gatctacgaa
     1501 cgcagtggca gcgccggaga gttcaagaag ttctgtttca ccgtgcgcaa gctgatcggg
     1561 tcaaatgacc tgccggagta cgatttgaag gaggaggcgg ggcaggctgg cccgatccta
     1621 gtcatgcgct accgcaacct gatcgagggc gaagcatccg ccggttccta atgtacggag
     1681 cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc gaaaaggact ctttcctgtg
     1741 gatagcacgt acattgggaa cccaaagccg tacattggga accggaaccc gtacattggg
     1801 aacccaaagc cgtacattgg gaaccggtca cacatgtaag tgactgatat aaaagagaaa
     1861 aaaggcgatt tttccgccta aaactcttta aaacttatta aaactcttaa aacccgcctg
     1921 gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc tgcaaaaagc gcctaccctt
     1981 cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc ctatcgcggc cgctggccgc
     2041 tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc gcggacaagc cgcgccgtcg
     2101 ccactcgacc gccggcgccc acatcaaggc accctgcctc gcgcgtttcg gtgatgacgg
     2161 tgaaaacctc tgacacatgc agctcccggt gacggtcaca gcttgtctgt aagcggatgc
     2221 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     2281 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     2341 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     2401 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2461 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2521 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2581 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2641 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2701 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2761 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2821 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2881 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2941 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3001 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     3061 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3121 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3181 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3241 tcacgttaag ggattttggt catgcattct aggtgattag aaaaactcat cgagcatcaa
     3301 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     3361 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     3421 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     3481 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     3541 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     3601 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagtc gaaatacgcg
     3661 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     3721 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     3781 tttccctggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     3841 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     3901 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     3961 atacaatcgg tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     4021 atataaatca gcatccatgt tggaatttaa tcgcggcctt gagcaagacg tttcccgttg
     4081 aatatggctc ataacagaac ttattatttc cttcctcttt tctacagtat ttaaagatac
     4141 cccaagaagc taattataac aagacgaact ccaattcact gttccttgca ttctaaaacc
     4201 ttaaatacca gaaaacagct ttttcaaagt tgttttcaaa gttggcgtat aacatagtat
     4261 cgacggagcc gattttgaaa ccgcggtgat cacaggcagc aacgctctgt catcgttaca
     4321 atcaacatgc taccctccgc gagatcatcc gtgtttcaaa cccggcagct tagttgccgt
     4381 tcttccgaat agcatcggta acatgagcaa agtctgccgc cttacaacgg ctctcccgct
     4441 gacgccgtcc cggactgatg ggctgcctgt atcgagtggt gattttgtgc cgagctgccg
     4501 gtcggggagc tgttggctgg ctggtggcag gatatattgt ggtgtaaaca taacgaattc
     4561 gtctcaggag actagagcca agctgatctc ctttgccccg gagatcacca tggacgactt
     4621 tctctatctc tacgatctag gaagaaagtt cgacggagaa ggtgacgata ccatgttcac
     4681 caccgataat gagaagatta gcctcttcaa tttcagaaag aatgctgacc cacagatggt
     4741 tagagaggcc tacgcggcag gtctgatcaa gacgatctac ccgagtaata atctccagga
     4801 gatcaaatac cttcccaaga aggttaaaga tgcagtcaaa agattcagga ctaactgcat
     4861 caagaacaca gagaaagata tatttctcaa gatcagaagt actattccag tatggacgat
     4921 tcaaggcttg cttcataaac caaggcaagt aatagagatt ggagtctcta agaaagtagt
     4981 tcctactgaa tcaaaggcca tggagtcaaa aattcagatc gaggatctaa cagaactcgc
     5041 cgtgaagact ggcgaacagt tcatacagag tcttttacga ctcaatgaca agaagaaaat
     5101 cttcgtcaac atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt
     5161 ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct
     5221 cggattccat tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg
     5281 cacctacaaa tgccatcatt gcgataaagg aaaggctatc gttcaagatg cccctgccga
     5341 cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc
     5401 aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc
     5461 acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga
     5521 gaggactccg gtatttttac aacaattacc acaacaaaac aaacaacaaa caacattaca
     5581 atttactatt ctagtcgaaa tgacaacaat gccccgcccc aagctcaagt ccgatgacga
     5641 ggtactcgag gccgccaccg tagtgctgaa gcgttgcggt cccatagagt tcacgctcag
     5701 cggagtagca aaggaggtgg ggctctcccg cgcagcgtta atccagcgct tcaccaaccg
     5761 cgatacgctg ctggtgagga tgatggagcg cggcgtcgag caggtgcggc attacctgaa
     5821 tgcgataccg ataggcgcag ggccgcaagg gctctgggaa tttttgcagg tgctcgttcg
     5881 gagcatgaac actcgcaacg acttctcggt gaactatctc atctcctggt acgagctcca
     5941 ggtgccggag ctacgcacgc ttgcgatcca gcggaaccgc gcggtggtgg aggggatccg
     6001 caagcgactg cccccaggtg ctcctgcggc agctgagttg ctcctgcact cggtcatcgc
     6061 tggcgcgacg atgcagtggg ccgtcgatcc ggatggtgag ctagctgatc atgtgctggc
     6121 tcagatcgct gccatcctgt gtttaatgtt tcccgaacac gacgatttcc aactcctcca
     6181 ggcacatgcg tccgcgtaca gcttaattaa cagtgctggt agtgctggta gtgctggtat
     6241 gatgttctta ccaaccgatt attgttgcag gttaagcgat caagagtata tggagctttt
     6301 gtttgagaat ggccagattc ttgcaaaggg ccaaagatcc aacgtttctc tgcataatca
     6361 acgtaccaaa tcgatcatgg atttgtatga ggcagagtat aacgaggatt tcatgaagag
     6421 tatcatccat ggtggtggtg gtgccatcac aaatctcggg gacacgcagg ttgttccaca
     6481 aagtcatgtt gctgctgccc atgaaacaaa catgttggaa agcaataaac atgttgaccc
     6541 aaagaagaag aggaaggtgt aggcttggaa tggatcttcg atcccgatcg ttcaaacatt
     6601 tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgacgat tatcatataa
     6661 tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg
     6721 agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa
     6781 atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg
     6841 gaattgccaa gctaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt
     6901 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     6961 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     7021 ataaccctga taaatgcttc aataatggga ccgactcgcg ctgtcatgag acgaattctg
     7081 acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat aatcggatat
     7141 ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc caaccacagg
     7201 gttcccctcg ggatcaaagt actttgatcc aacccctccg ctgctatagt gcagtcggct
     7261 tctgacgttc agtgcagccg tcatctgaaa acgacatgtc gcacaagtcc taagttacgc
     7321 gacaggctgc cgccctgccc ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa
     7381 agtagaatac ttgcgactag aaccggagac attacgccat gaacaagagc gccgccgctg
     7441 gcctgctggg ctatgcccgc gtcagcaccg acgaccagga cttgaccaac caacgggccg
     7501 aactgcacgc ggccggctgc accaagctgt tttccgagaa gatcaccggc accaggcgcg
     7561 accgcccgga gctggccagg atgcttgacc acctacgccc tggcgacgtt gtgacagtga
     7621 ccaggctaga ccgcctggcc cgcagcaccc gcgacctact ggacattgcc gagcgcatcc
     7681 aggaggccgg cgcgggcctg cgtagcctgg cagagccgtg ggccgacacc accacgccgg
     7741 ccggccgcat ggtgttgacc gtgttcgccg gcattgccga gttcgagcgt tccctaatca
     7801 tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc
     7861 gccctaccct caccccggca cagatcgcgc acgcccgcga gctgatcgac caggaaggcc
     7921 gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac
     7981 ttgagcgcag cgaggaagtg acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg
     8041 acgcattgac cgaggccgac gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag
     8101 catgaaaccg caccaggacg gccaggacga accgtttttc attaccgaag agatcgaggc
     8161 ggagatgatc gcggccgggt acgtgttcga gccgcccgcg cacctctcaa ccgtgcggct
     8221 gcatgaaatc ctggccggtt tgtctgatgc caagctggcg gcctggccgg ccagcttggc
     8281 cgctgaagaa accgagcgcc gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt
     8341 gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat aaacaaatac gcaaggggaa
     8401 cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt caggcaagac gaccatcgca
     8461 acccatctag cccgcgccct gcaactcgcc ggggccgatg ttctgttagt cgattccgat
     8521 ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc
     8581 ggcatcgacc gcccgacgat tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta
     8641 gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac
     8701 ttcgtgctga ttccggtgca gccaagccct tacgacatat gggccaccgc cgacctggtg
     8761 gagctggtta agcagcgcat tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg
     8821 tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg ccga
//
