LOCUS       sequence-450185-        6550 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses an sgRNA targeting the NbCysP6 gene in Nicotiana 
            benthamiana with an Arabidopsis U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6550)
  AUTHORS   Singh AA, Pillay P, Naicker P, Alexandre K, Malatji K, Mach L, 
            Steinkellner H, Vorster J, Chikwamba R, Tsekoa TL
  TITLE     Transient proteolysis reduction of Nicotiana benthamiana-produced 
            CAP256 broadly neutralizing antibodies using CRISPR/Cas9.
  JOURNAL   Front Plant Sci. 2022 Aug 18;13:953654. doi: 
            10.3389/fpls.2022.953654. eCollection 2022.
  PUBMED    36061808
REFERENCE   2  (bases 1 to 6550)
  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: "Front Plant
            Sci."; date: "2022-08-18"; volume: "13:953654. doi"; pages: " 
            10.3389/fpls.2022.953654. eCollection 2022"
COMMENT     Sequence Label: pICH86966::AtU6p::sgRNA:NbCysP6
FEATURES             Location/Qualifiers
     source          1..6550
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    32..56
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(179..767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(259..278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(938..1732)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCAKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     promoter        complement(1733..1837)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2120..2314
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(2380..3453)
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGASSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     CDS             complement(3882..4511)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     misc_feature    4775..4799
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        4976..5155
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     promoter        5008..5191
                     /function="NOS promoter"
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     CDS             5212..6006
                     /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(5266..5285)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5876..5895
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      6032..6284
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     promoter        6311..6385
                     /label=AtU6 promoter
                     /note="consensus promoter from the three Arabidopsis U6 
                     snRNA genes (Waibel and Filipowicz, 1990; Nekrasov et al., 
                     2013)"
     misc_RNA        6406..6481
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(6493..6517)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 gggacacgaa gtggtttaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa
       61 gagaaaagag cgtttattag aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg
      121 ttcgtccatt tgtatgtgcc aaccacagga accgtaaaaa ggccgcgttg ctggcgtttt
      181 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
      241 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
      301 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
      361 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
      421 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
      481 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
      541 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
      601 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
      661 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
      721 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
      781 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
      841 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
      901 caatctaaag tatatatgag taaacttggt ctgacagcta aaacaattca tccagtaaaa
      961 tataatattt tattttctcc caatcaggct tgatccccag taagtcaaaa aatagctcga
     1021 catactgttc ttccccgata tcctccctga tcgaccggac gcagaaggca atgtcatacc
     1081 acttgtccgc cctgccgctt ctcccaagat caataaagcc acttactttg ccatctttca
     1141 caaagatgtt gctgtctccc aggtcgccgt gggaaaagac aagttcctct tcgggctttt
     1201 ccgtctttaa aaaatcatac agctcgcgcg gatctttaaa tggagtgtcc tcttcccagt
     1261 tttcgcaatc cacatcggcc agatcgttat tcagtaagta atccaattcg gctaagcggc
     1321 tgtctaagct attcgtatag ggacaatccg atatgtcgat ggagtgaaag agcctgatgc
     1381 actccgcata cagctcgata atcttttcag ggctttgttc atcttcatac tcttccgagc
     1441 aaaggacgcc atcggcctca ctcatgagca gattgctcca gccatcatgc cgttcaaagt
     1501 gcaggacctt tggaacaggc agctttcctt ccagccatag catcatgtcc ttttcccgtt
     1561 ccacatcata ggtggtccct ttataccggc tgtccgtcat ttttaaatat aggttttcat
     1621 tttctcccac cagcttatat accttagcag gagacattcc ttccgtatct tttgcgcagc
     1681 ggtatttttc gatcagtttt ttcaattccg gtgatattct cattttagcc atactcttcc
     1741 tttttcaata ttattgaagc atttatcaag gttattgtct catgagcgga tacatatttg
     1801 aatgtattta gaaaaataaa caaatagggg ttccgcgcac gaattggcca gcgctgccat
     1861 ttttggggtg aggccgttcg cggccgaggg gcgcagcccc tggggggatg ggagctcggt
     1921 gccttgatgt gggcgccggc ggtcgagtgg cgacggcgcg gcttgtccgc gccctggtag
     1981 attgcctggc cgtaggccag ccatttttga gcggccagcg gccgcgatag gccgacgcga
     2041 agcggcgggg cgtagggagc gcagcgaccg aagggtaggc gctttttgca gctcttcggc
     2101 tgtgcgctgg ccagacagtt atgcacaggc caggcgggtt ttaagagttt taataagttt
     2161 taaagagttt taggcggaaa aatcgccttt tttctctttt atatcagtca cttacatgtg
     2221 tgaccggttc ccaatgtacg gctttgggtt cccaatgtac gggttccggt tcctaatgta
     2281 cggctttggg ttcccaatgt acgtgctatc cacaggaaag aagctttttc gacctttttc
     2341 ccctgctagg gcaatttgcc ctagcatctg ctccgtacat taggaaccgg cggatgcttc
     2401 gccctcgatc aggttgcggt agcgcatgac taggatcggg ccagcctgcc ccgcctcctc
     2461 cttcaaatcg tactccggca ggtcatttga cccgatcagc ttgcgcacgg tgaaacagaa
     2521 cttcttgaac tctccggcgc tgccactgcg ttcgtagatc gtcttgaaca accatctggc
     2581 ttctgccttg cctgcggcgc ggcgtgccag gcggtagaga aaacggccga tgccgggatc
     2641 gatcaaaaag taatcggggt gaaccgtcag cacgtccggg ttcttgcctt ctgtgatctc
     2701 gcggtacatc caatcagcta gctcgatctc gatgtactcc ggccgcccgg tttcgctctt
     2761 tacgatcttg tagcggctaa tcaaggcttc accctcggat accgtcacca ggcggccgtt
     2821 cttggccttc ttcgtacgct gcatggcaac gtgcgtggtg tttaaccgaa tgcaggtttc
     2881 taccaggtcg tctttctgct ttccgccatc ggctcgccgg cagaacttga gtacgtccgc
     2941 aacgtgtgga cggaacacgc ggccgggctt gtctcccttc ccttcccggt atcggttcat
     3001 ggattcggtt agatgggaaa ccgccatcag taccaggtcg taatcccaca cactggccat
     3061 gccggccggc cctgcggaaa cctctacgtg cccgtctgga agctcgtagc ggatcacctc
     3121 gccagctcgt cggtcacgct tcgacagacg gaaaacggcc acgtccatga tgctgcgact
     3181 atcgcgggtg cccacgtcat agagcatcgg aacgaaaaaa tctggttgct cgtcgccctt
     3241 gggcggcttc ctaatcgacg acgcaccggc tgccggcggt tgccgggatt ctttgcggat
     3301 tcgatcagcg gccgcttgcc acgattcacc ggggcgtgct tctgcctcga tgcgttgccg
     3361 ctgggcggcc tgcgcggcct tcaacttctc caccaggtca tcacccagcg ccgcgccgat
     3421 ttgtaccggg ccggatggtt tgcgaccgct cacgccgatt cctcgggctt gggggttcca
     3481 gtgccattgc agggccggca gacaacccag ccgcttacgc ctggccaacc gcccgttcct
     3541 ccacacatgg ggcattccac ggcgtcggtg cctggttgtt cttgattttc catgccgcct
     3601 cctttagccg ctaaaattca tctactcatt tattcatttg ctcatttact ctggtagctg
     3661 cgcgatgtat tcagatagca gctcggtaat ggtcttgcct tggcgtaccg cgtacatctt
     3721 cagcttggtg tgatcctccg ccggcaactg aaagttgacc cgcttcatgg ctggcgtgtc
     3781 tgccaggctg gccaacgttg cagccttgct gctgcgtgcg ctcggacggc cggcacttag
     3841 cgtgtttgtg cttttgctca ttttctcttt acctcattaa ctcaaatgag ttttgattta
     3901 atttcagcgg ccagcgcctg gacctcgcgg gcagcgtcgc cctcgggttc tgattcaaga
     3961 acggttgtgc cggcggcggc agtgcctggg tagctcacgc gctgcgtgat acgggactca
     4021 agaatgggca gctcgtaccc ggccagcgcc tcggcaacct caccgccgat gcgcgtgcct
     4081 ttgatcgccc gcgacacgac aaaggccgct tgtagccttc catccgtgac ctcaatgcgc
     4141 tgcttaacca gctccaccag gtcggcggtg gcccatatgt cgtaagggct tggctgcacc
     4201 ggaatcagca cgaagtcggc tgccttgatc gcggacacag ccaagtccgc cgcctggggc
     4261 gctccgtcga tcactacgaa gtcgcgccgg ccgatggcct tcacgtcgcg gtcaatcgtc
     4321 gggcggtcga tgccgacaac ggttagcggt tgatcttccc gcacggccgc ccaatcgcgg
     4381 gcactgccct ggggatcgga atcgactaac agaacatcgg ccccggcgag ttgcagggcg
     4441 cgggctagat gggttgcgat ggtcgtcttg cctgacccgc ctttctggtt aagtacagcg
     4501 ataaccttca tgcgttcccc ttgcgtattt gtttatttac tcatcgcatc atatacgcag
     4561 cgaccgcatg acgcaagctg ttttactcaa atacacatca cctttttaga cggcggcgct
     4621 cggtttcttc agcggccaag ctggccggcc aggccgccag cttggcatca gacaaaccgg
     4681 ccaggatttc atgcagccgc acggctgatg ggctgcctgt atcgagtggt gattttgtgc
     4741 cgagctgccg gtcggggagc tgttggctgg ctggtggcag gatatattgt ggtgtaaaca
     4801 aattgacgct tagacaactt aataacacat tgcggacgtt tttaatgtac tggggtggat
     4861 gcagtgggcc ccactctgtg tgccgaattc ggatccggag agcggagaat taagggagtc
     4921 acgttatgac cccgccgatg acgcgggaca agccgtttta cgtttggaac tgacagaacc
     4981 gcaacgttga aggagccact cagccgcggg tttctggagt ttaatgagct aagcacatac
     5041 gtcagaaacc attattgcgc gttcaaaagt cgcctaaggt cactatcagc tagcaaatat
     5101 ttcttgtcaa aaatgctcca ctgacgttcc ataaattccc ctcggtatcc aattagagtc
     5161 tcatattcac tctcaatcca aataatctgc accggatctg gatcgtttcg catgattgaa
     5221 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     5281 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     5341 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact ccaggacgag
     5401 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     5461 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     5521 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     5581 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     5641 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     5701 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gtatgcccga cggcgaggat
     5761 ctcgtcgtga ctcatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     5821 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     5881 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     5941 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     6001 ttctgagcgg gtctggatct agagtcaagc agatcgttca aacatttggc aataaagttt
     6061 cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta
     6121 cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat
     6181 gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa
     6241 ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgacgct gcaagaattc
     6301 aagcttggag tgatcaaaag tcccacatcg atcaggtgat atatagcagc ttagtttata
     6361 taatgataga gtcgacatag cgattgagga agactaccct tacacgtttt agagctagaa
     6421 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     6481 ctttttttct agacccagct ttcttgtaca aagttggcat tacgctacta gaggatgcac
     6541 atgtgaccga
//
