LOCUS       sequence-427149-        8186 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TU for SV40-AcrIIA4 protein Codon optimized for N. benthamiana 
            expression under the regulation of the 35S promoter and Tnos 
            terminator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8186)
  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 8186)
  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: pDGB3_alpha1 P35S:SV40-AcrIIA4_CONb:Tnos (GB3344)
FEATURES             Location/Qualifiers
     source          1..8186
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    17..41
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             1342..1971
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             2400..3473
                     /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      3539..3733
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(3969..3991)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4077..4217
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4132..4151
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4232..4249)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4403..4991)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4483..4502)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5079..5894)
                     /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(5079..5894)
                     /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     5802..5821
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    6327..6351
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        6984..7328
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     7272..7292
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             7405..7425
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      7710..7962
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(7997..8015)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gtcatgagac gaattctgac aggatatatt ggcgggtaaa cctaagagaa aagagcgttt
       61 attagaataa tcggatattt aaaagggcgt gaaaaggttt atccgttcgt ccatttgtat
      121 gtgcatgcca accacagggt tcccctcggg atcaaagtac tttgatccaa cccctccgct
      181 gctatagtgc agtcggcttc tgacgttcag tgcagccgtc atctgaaaac gacatgtcgc
      241 acaagtccta agttacgcga caggctgccg ccctgccctt ttcctggcgt tttcttgtcg
      301 cgtgttttag tcgcataaag tagaatactt gcgactagaa ccggagacat tacgccatga
      361 acaagagcgc cgccgctggc ctgctgggct atgcccgcgt cagcaccgac gaccaggact
      421 tgaccaacca acgggccgaa ctgcacgcgg ccggctgcac caagctgttt tccgagaaga
      481 tcaccggcac caggcgcgac cgcccggagc tggccaggat gcttgaccac ctacgccctg
      541 gcgacgttgt gacagtgacc aggctagacc gcctggcccg cagcacccgc gacctactgg
      601 acattgccga gcgcatccag gaggccggcg cgggcctgcg tagcctggca gagccgtggg
      661 ccgacaccac cacgccggcc ggccgcatgg tgttgaccgt gttcgccggc attgccgagt
      721 tcgagcgttc cctaatcatc gaccgcaccc ggagcgggcg cgaggccgcc aaggcccgag
      781 gcgtgaagtt tggcccccgc cctaccctca ccccggcaca gatcgcgcac gcccgcgagc
      841 tgatcgacca ggaaggccgc accgtgaaag aggcggctgc actgcttggc gtgcatcgct
      901 cgaccctgta ccgcgcactt gagcgcagcg aggaagtgac gcccaccgag gccaggcggc
      961 gcggtgcctt ccgtgaggac gcattgaccg aggccgacgc cctggcggcc gccgagaatg
     1021 aacgccaaga ggaacaagca tgaaaccgca ccaggacggc caggacgaac cgtttttcat
     1081 taccgaagag atcgaggcgg agatgatcgc ggccgggtac gtgttcgagc cgcccgcgca
     1141 cctctcaacc gtgcggctgc atgaaatcct ggccggtttg tctgatgcca agctggcggc
     1201 ctggccggcc agcttggccg ctgaagaaac cgagcgccgc cgtctaaaaa ggtgatgtgt
     1261 atttgagtaa aacagcttgc gtcatgcggt cgctgcgtat atgatgcgat gagtaaataa
     1321 acaaatacgc aaggggaacg catgaaggtt atcgctgtac ttaaccagaa aggcgggtca
     1381 ggcaagacga ccatcgcaac ccatctagcc cgcgccctgc aactcgccgg ggccgatgtt
     1441 ctgttagtcg attccgatcc ccagggcagt gcccgcgatt gggcggccgt gcgggaagat
     1501 caaccgctaa ccgttgtcgg catcgaccgc ccgacgattg accgcgacgt gaaggccatc
     1561 ggccggcgcg acttcgtagt gatcgacgga gcgccccagg cggcggactt ggctgtgtcc
     1621 gcgatcaagg cagccgactt cgtgctgatt ccggtgcagc caagccctta cgacatatgg
     1681 gccaccgccg acctggtgga gctggttaag cagcgcattg aggtcacgga tggaaggcta
     1741 caagcggcct ttgtcgtgtc gcgggcgatc aaaggcacgc gcatcggcgg tgaggttgcc
     1801 gaggcgctgg ccgggtacga gctgcccatt cttgagtccc gtatcacgca gcgcgtgagc
     1861 tacccaggca ctgccgccgc cggcacaacc gttcttgaat cagaacccga gggcgacgct
     1921 gcccgcgagg tccaggcgct ggccgctgaa attaaatcaa aactcatttg agttaatgag
     1981 gtaaagagaa aatgagcaaa agcacaaaca cgctaagtgc cggccgtccg agcgcacgca
     2041 gcagcaaggc tgcaacgttg gccagcctgg cagacacgcc agccatgaag cgggtcaact
     2101 ttcagttgcc ggcggaggat cacaccaagc tgaagatgta cgcggtacgc caaggcaaga
     2161 ccattaccga gctgctatct gaatacatcg cgcagctacc agagtaaatg agcaaatgaa
     2221 taaatgagta gatgaatttt agcggctaaa ggaggcggca tggaaaatca agaacaacca
     2281 ggcaccgacg ccgtggaatg ccccatgtgt ggaggaacgg gcggttggcc aggcgtaagc
     2341 ggctgggttg cctgccggcc ctgcaatggc actggaaccc ccaagcccga ggaatcggcg
     2401 tgagcggtcg caaaccatcc ggcccggtac aaatcggcgc ggcgctgggt gatgacctgg
     2461 tggagaagtt gaaggccgcg caggccgccc agcggcaacg catcgaggca gaagcacgcc
     2521 ccggtgaatc gtggcaagcg gccgctgatc gaatccgcaa agaatcccgg caaccgccgg
     2581 cagccggtgc gccgtcgatt aggaagccgc ccaagggcga cgagcaacca gattttttcg
     2641 ttccgatgct ctatgacgtg ggcacccgcg atagtcgcag catcatggac gtggccgttt
     2701 tccgtctgtc gaagcgtgac cgacgagctg gcgaggtgat ccgctacgag cttccagacg
     2761 ggcacgtaga ggtttccgca gggccggccg gcatggccag tgtgtgggat tacgacctgg
     2821 tactgatggc ggtttcccat ctaaccgaat ccatgaaccg ataccgggaa gggaagggag
     2881 acaagcccgg ccgcgtgttc cgtccacacg ttgcggacgt actcaagttc tgccggcgag
     2941 ccgatggcgg aaagcagaaa gacgacctgg tagaaacctg cattcggtta aacaccacgc
     3001 acgttgccat gcagcgtacg aagaaggcca agaacggccg cctggtgacg gtatccgagg
     3061 gtgaagcctt gattagccgc tacaagatcg taaagagcga aaccgggcgg ccggagtaca
     3121 tcgagatcga gctagctgat tggatgtacc gcgagatcac agaaggcaag aacccggacg
     3181 tgctgacggt tcaccccgat tactttttga tcgatcccgg catcggccgt tttctctacc
     3241 gcctggcacg ccgcgccgca ggcaaggcag aagccagatg gttgttcaag acgatctacg
     3301 aacgcagtgg cagcgccgga gagttcaaga agttctgttt caccgtgcgc aagctgatcg
     3361 ggtcaaatga cctgccggag tacgatttga aggaggaggc ggggcaggct ggcccgatcc
     3421 tagtcatgcg ctaccgcaac ctgatcgagg gcgaagcatc cgccggttcc taatgtacgg
     3481 agcagatgct agggcaaatt gccctagcag gggaaaaagg tcgaaaagga ctctttcctg
     3541 tggatagcac gtacattggg aacccaaagc cgtacattgg gaaccggaac ccgtacattg
     3601 ggaacccaaa gccgtacatt gggaaccggt cacacatgta agtgactgat ataaaagaga
     3661 aaaaaggcga tttttccgcc taaaactctt taaaacttat taaaactctt aaaacccgcc
     3721 tggcctgtgc ataactgtct ggccagcgca cagccgaaga gctgcaaaaa gcgcctaccc
     3781 ttcggtcgct gcgctcccta cgccccgccg cttcgcgtcg gcctatcgcg gccgctggcc
     3841 gctcaaaaat ggctggccta cggccaggca atctaccagg gcgcggacaa gccgcgccgt
     3901 cgccactcga ccgccggcgc ccacatcaag gcaccctgcc tcgcgcgttt cggtgatgac
     3961 ggtgaaaacc tctgacacat gcagctcccg gtgacggtca cagcttgtct gtaagcggat
     4021 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
     4081 gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag
     4141 agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga
     4201 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4261 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4321 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4381 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4441 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4501 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4561 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4621 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4681 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4741 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4801 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4861 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4921 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4981 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5041 actcacgtta agggattttg gtcatgcatt ctaggtgatt agaaaaactc atcgagcatc
     5101 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     5161 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     5221 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     5281 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     5341 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     5401 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag tcgaaatacg
     5461 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     5521 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     5581 gttttccctg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     5641 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     5701 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     5761 ccatacaatc ggtagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     5821 ccatataaat cagcatccat gttggaattt aatcgcggcc ttgagcaaga cgtttcccgt
     5881 tgaatatggc tcataacaga acttattatt tccttcctct tttctacagt atttaaagat
     5941 accccaagaa gctaattata acaagacgaa ctccaattca ctgttccttg cattctaaaa
     6001 ccttaaatac cagaaaacag ctttttcaaa gttgttttca aagttggcgt ataacatagt
     6061 atcgacggag ccgattttga aaccgcggtg atcacaggca gcaacgctct gtcatcgtta
     6121 caatcaacat gctaccctcc gcgagatcat ccgtgtttca aacccggcag cttagttgcc
     6181 gttcttccga atagcatcgg taacatgagc aaagtctgcc gccttacaac ggctctcccg
     6241 ctgacgccgt cccggactga tgggctgcct gtatcgagtg gtgattttgt gccgagctgc
     6301 cggtcgggga gctgttggct ggctggtggc aggatatatt gtggtgtaaa cataacgaat
     6361 tcgtctcagg agactagagc caagctgatc tcctttgccc cggagatcac catggacgac
     6421 tttctctatc tctacgatct aggaagaaag ttcgacggag aaggtgacga taccatgttc
     6481 accaccgata atgagaagat tagcctcttc aatttcagaa agaatgctga cccacagatg
     6541 gttagagagg cctacgcggc aggtctgatc aagacgatct acccgagtaa taatctccag
     6601 gagatcaaat accttcccaa gaaggttaaa gatgcagtca aaagattcag gactaactgc
     6661 atcaagaaca cagagaaaga tatatttctc aagatcagaa gtactattcc agtatggacg
     6721 attcaaggct tgcttcataa accaaggcaa gtaatagaga ttggagtctc taagaaagta
     6781 gttcctactg aatcaaaggc catggagtca aaaattcaga tcgaggatct aacagaactc
     6841 gccgtgaaga ctggcgaaca gttcatacag agtcttttac gactcaatga caagaagaaa
     6901 atcttcgtca acatggtgga gcacgacact ctcgtctact ccaagaatat caaagataca
     6961 gtctcagaag accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc
     7021 ctcggattcc attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt
     7081 ggcacctaca aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcccctgcc
     7141 gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt
     7201 ccaaccacgt cttcaaagca agtggattga tgtgatatct ccactgacgt aagggatgac
     7261 gcacaatccc actatccttc gcaagaccct tcctctatat aaggaagttc atttcatttg
     7321 gagaggactc cggtattttt acaacaatta ccacaacaaa acaaacaaca aacaacatta
     7381 caatttacta ttctagtcga aatgcctaag aaaaaacgaa aggtcaacat taatgagctt
     7441 attcgtgaaa ttaaaaacaa agattatacg gctaagttga gtgggacaga cagcaacagt
     7501 ataactcaac ttatcataca cgtcaataac gacgggaacg agtacgtgat tagcgaatct
     7561 gaaaacgagt caattgtgga gaagttcatt tctgcattta agtcaggttg gaatcaggag
     7621 tatgaagatg aggaagaatt ttataacgat atgcagacca taactctgaa gtccgaactg
     7681 aattgagctt ggaatggatc ttcgatcccg atcgttcaaa catttggcaa taaagtttct
     7741 taagattgaa tcctgttgcc ggtcttgcga cgattatcat ataatttctg ttgaattacg
     7801 ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga
     7861 ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact
     7921 aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcgggaattg ccaagctaat
     7981 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     8041 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     8101 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     8161 cttcaataat gggaccgact cgcgct
//
