LOCUS       sequence-490469-        8881 bp    DNA     circular SYN 23-OCT-2025
DEFINITION  Hsp18.2 (heat inducible) promoter driving the expression of sgRNA-B.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8881)
  AUTHORS   Khan MA, Herring G, Zhu JY, Oliva M, Fourie E, Johnston B, Zhang Z, 
            Potter J, Pineda L, Pflueger J, Swain T, Pflueger C, Lloyd JPB, 
            Secco D, Small I, Kidd BN, Lister R
  TITLE     CRISPRi-based circuits to control gene expression in plants.
  JOURNAL   Nat Biotechnol. 2025 Mar;43(3):416-430. doi: 
            10.1038/s41587-024-02236-w. Epub 2024 May 20.
  PUBMED    38769424
REFERENCE   2  (bases 1 to 8881)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 23, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41587-024-02236-w"; journalName: "Nat Biotechnol"; date: 
            "2025-03"; volume: "43"; issue: "3"; pages: "416-430"
COMMENT     Sequence Label: LLP1014
FEATURES             Location/Qualifiers
     source          1..8881
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    433..457
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    1302..1321
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        1341..1416
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1417..1436
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     polyA_signal    1471..1645
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    1666..1690
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             2991..3620
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             4049..5122
                     /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      5188..5382
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(5618..5640)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5726..5866
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5781..5800
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5881..5898)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6052..6640)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6132..6151)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(8066..8881)
                     /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(8066..8881)
                     /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     8789..8808
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 aacagaactt attatttcct tcctcttttc tacagtattt aaagataccc caagaagcta
       61 attataacaa gacgaactcc aattcactgt tccttgcatt ctaaaacctt aaataccaga
      121 aaacagcttt ttcaaagttg ttttcaaagt tggcgtataa catagtatcg acggagccga
      181 ttttgaaacc gcggtgatca caggcagcaa cgctctgtca tcgttacaat caacatgcta
      241 ccctccgcga gatcatccgt gtttcaaacc cggcagctta gttgccgttc ttccgaatag
      301 catcggtaac atgagcaaag tctgccgcct tacaacggct ctcccgctga cgccgtcccg
      361 gactgatggg ctgcctgtat cgagtggtga ttttgtgccg agctgccggt cggggagctg
      421 ttggctggct ggtggcagga tatattgtgg tgtaaacata acgaattgct cttcagcaag
      481 gagcatgcaa gttctattga ttagaaacca aaaccatgac atatcacatt ttcaattttt
      541 aaccccaagt ttcccaaaaa ggtaatcaca attactttcc atggtcattt cttctggttc
      601 aagcatgaca tgaacaggca ataaataagt tgagattttg atcacagtaa ctgatacttg
      661 aatcgaatca tttagatttt tttttttttt agtttacttg tttagtaaat atgttgtcta
      721 tgtttgtcac aaaaacgtgg ctcagttctt gtatatatgg agacaaaaaa atccattaaa
      781 agattgttga cattctcgga aatttagtgc caactgttat tgcgagaact tactatagtt
      841 ttcctttggc gaaaagctaa taatcttaaa tcttgatttt gtcctctttt ctctgagtta
      901 gattttctta aattccactt ccgacctatt aagaaatggg cttttgcaaa gaagatccgc
      961 ttcactgagc ccgtatctcg aagaggataa tacaacaaca aagcaaaacg gcacgtagtt
     1021 ttaattgtaa ccaaggattg catttcggtc ttgtttcaac aaacgaaact tcctgaaatg
     1081 ccaagaaaaa tctggtcatt tcaccacagt gatcattgtg tatgtgttct aaagactcca
     1141 agcgaaggtt ttagaaaaag gagcattttc tattctattc aagaaactcg aagaacattc
     1201 tctcttcatc ctctaacttc cctataaata tgtcctttgc taatcagatc aaatcagcag
     1261 gaaaatcaag aaccaaaagt ctcccgaaaa gcaacgatac tgttcactgc cgtataggca
     1321 gtacctcatc aggaacatgt gttttagagc tagaaatagc aagttaaaat aaggctagtc
     1381 cgttatcaac ttgaaaaagt ggcaccgagt cggtgcgttc actgccgtat aggcaggctt
     1441 ctctagctag agtcgatcga caagctcgag tttctccata ataatgtgtg agtagttccc
     1501 agataaggga attagggttc ctatagggtt tcgctcatgt gttgagcata taagaaaccc
     1561 ttagtatgta tttgtatttg taaaatactt ctatcaataa aatttctaat tcctaaaacc
     1621 aaaatccagt actaaaatcc agatcgctat actgaagagc aattctgaca ggatatattg
     1681 gcgggtaaac ctaagagaaa agagcgttta ttagaataat cggatattta aaagggcgtg
     1741 aaaaggttta tccgttcgtc catttgtatg tgcatgccaa ccacagggtt cccctcggga
     1801 tcaaagtact ttgatccaac ccctccgctg ctatagtgca gtcggcttct gacgttcagt
     1861 gcagccgtca tctgaaaacg acatgtcgca caagtcctaa gttacgcgac aggctgccgc
     1921 cctgcccttt tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt agaatacttg
     1981 cgactagaac cggagacatt acgccatgaa caagagcgcc gccgctggcc tgctgggcta
     2041 tgcccgcgtc agcaccgacg accaggactt gaccaaccaa cgggccgaac tgcacgcggc
     2101 cggctgcacc aagctgtttt ccgagaagat caccggcacc aggcgcgacc gcccggagct
     2161 ggccaggatg cttgaccacc tacgccctgg cgacgttgtg acagtgacca ggctagaccg
     2221 cctggcccgc agcacccgcg acctactgga cattgccgag cgcatccagg aggccggcgc
     2281 gggcctgcgt agcctggcag agccgtgggc cgacaccacc acgccggccg gccgcatggt
     2341 gttgaccgtg ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg
     2401 gagcgggcgc gaggccgcca aggcccgagg cgtgaagttt ggcccccgcc ctaccctcac
     2461 cccggcacag atcgcgcacg cccgcgagct gatcgaccag gaaggccgca ccgtgaaaga
     2521 ggcggctgca ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga
     2581 ggaagtgacg cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga
     2641 ggccgacgcc ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac
     2701 caggacggcc aggacgaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg
     2761 gccgggtacg tgttcgagcc gcccgcgcac ctctcaaccg tgcggctgca tgaaatcctg
     2821 gccggtttgt ctgatgccaa gctggcggcc tggccggcca gcttggccgc tgaagaaacc
     2881 gagcgccgcc gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc
     2941 gctgcgtata tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta
     3001 tcgctgtact taaccagaaa ggcgggtcag gcaagacgac catcgcaacc catctagccc
     3061 gcgccctgca actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg
     3121 cccgcgattg ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc
     3181 cgacgattga ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg atcgacggag
     3241 cgccccaggc ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc
     3301 cggtgcagcc aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc
     3361 agcgcattga ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca
     3421 aaggcacgcg catcggcggt gaggttgccg aggcgctggc cgggtacgag ctgcccattc
     3481 ttgagtcccg tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg
     3541 ttcttgaatc agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg gccgctgaaa
     3601 ttaaatcaaa actcatttga gttaatgagg taaagagaaa atgagcaaaa gcacaaacac
     3661 gctaagtgcc ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc
     3721 agacacgcca gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct
     3781 gaagatgtac gcggtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc
     3841 gcagctacca gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag
     3901 gaggcggcat ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg
     3961 gaggaacggg cggttggcca ggcgtaagcg gctgggttgc ctgccggccc tgcaatggca
     4021 ctggaacccc caagcccgag gaatcggcgt gagcggtcgc aaaccatccg gcccggtaca
     4081 aatcggcgcg gcgctgggtg atgacctggt ggagaagttg aaggccgcgc aggccgccca
     4141 gcggcaacgc atcgaggcag aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg
     4201 aatccgcaaa gaatcccggc aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc
     4261 caagggcgac gagcaaccag attttttcgt tccgatgctc tatgacgtgg gcacccgcga
     4321 tagtcgcagc atcatggacg tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg
     4381 cgaggtgatc cgctacgagc ttccagacgg gcacgtagag gtttccgcag ggccggccgg
     4441 catggccagt gtgtgggatt acgacctggt actgatggcg gtttcccatc taaccgaatc
     4501 catgaaccga taccgggaag ggaagggaga caagcccggc cgcgtgttcc gtccacacgt
     4561 tgcggacgta ctcaagttct gccggcgagc cgatggcgga aagcagaaag acgacctggt
     4621 agaaacctgc attcggttaa acaccacgca cgttgccatg cagcgtacga agaaggccaa
     4681 gaacggccgc ctggtgacgg tatccgaggg tgaagccttg attagccgct acaagatcgt
     4741 aaagagcgaa accgggcggc cggagtacat cgagatcgag ctagctgatt ggatgtaccg
     4801 cgagatcaca gaaggcaaga acccggacgt gctgacggtt caccccgatt actttttgat
     4861 cgatcccggc atcggccgtt ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga
     4921 agccagatgg ttgttcaaga cgatctacga acgcagtggc agcgccggag agttcaagaa
     4981 gttctgtttc accgtgcgca agctgatcgg gtcaaatgac ctgccggagt acgatttgaa
     5041 ggaggaggcg gggcaggctg gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg
     5101 cgaagcatcc gccggttcct aatgtacgga gcagatgcta gggcaaattg ccctagcagg
     5161 ggaaaaaggt cgaaaaggac tctttcctgt ggatagcacg tacattggga acccaaagcc
     5221 gtacattggg aaccggaacc cgtacattgg gaacccaaag ccgtacattg ggaaccggtc
     5281 acacatgtaa gtgactgata taaaagagaa aaaaggcgat ttttccgcct aaaactcttt
     5341 aaaacttatt aaaactctta aaacccgcct ggcctgtgca taactgtctg gccagcgcac
     5401 agccgaagtg ctgcaaaaag cgcctaccct tcggtcgctg cgctccctac gccccgccgc
     5461 ttcgcgtcgg cctatcgcgg ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa
     5521 tctaccaggg cgcggacaag ccgcgccgtc gccactcgac cgccggcgcc cacatcaagg
     5581 caccctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     5641 tgacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     5701 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     5761 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     5821 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct catccgcttc
     5881 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5941 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     6001 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     6061 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     6121 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     6181 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     6241 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     6301 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     6361 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6421 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6481 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6541 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6601 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6661 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggctgatga atcccctaat
     6721 gatttttatc aaaatcatta agttaaggta gatacacatc ttgtcatatg atcaaatggt
     6781 ttcgccaaaa atcaataatc agacaacaaa atgtgcgaac tcgatatttt acacgactct
     6841 ctttaccaat tctgccccga attacactta aaacgactca acagcttaac gttggcttgc
     6901 cacgccttac ttgactgtaa aactctcact cttaccgaac ttggccgtaa cctgccaacc
     6961 aaagcgagaa caaaacataa catcaaacga atcgaccgat tgttaggtaa tcgtcacctc
     7021 cacaaagagc gactcgctgt ataccgttgg catgctagct ttatctgttc gggcaatacg
     7081 atgcccattg tacttgttga ctggtctgat atccgtgagc aaaaacggct tatggtattg
     7141 cgagcttcag tcgcactaca cggtcgttct gttactcttt atgagaaagc gttcccgctt
     7201 tcagagcaat gttcaaagaa agctcatgac caatttctag ccgaccttgc gagcattcta
     7261 ccgagtaaca ccacaccgct cattgtcagt gatgctggct ttaaagtgcc atggtataaa
     7321 tccgttgaga agctgggttg gtactggtta agtcgagtaa gaggaaaagt acaatatgca
     7381 gacctaggag cggaaaactg gaaacctatc agcaacttac atgatatgtc atctagtcac
     7441 tcaaagactt taggctataa gaggctgact aaaagcaatc caatctcatg ccaaattcta
     7501 ttgtataaat ctcgctctaa aggccgaaaa aatcagcgct cgacacggac tcattgtcac
     7561 cacccgtcac ctaaaatcta ctcagcgtcg gcaaaggagc catggattct agcaactaac
     7621 ttacctgttg aaattcgaac acccaaacaa cttgttaata tctattcgaa gcgaatgcag
     7681 attgaagaaa ccttccgaga cttgaaaagt cctgcctacg gactaggcct acgccatagc
     7741 cgaacgagca gctcagagcg ttttgatatc atgctgctaa tcgccctgat gcttcaacta
     7801 acatgttggc ttgcgggcgt tcatgctcag aaacaaggtt gggacaagca cttccaggct
     7861 aacacagtca gaaatcgaaa cgtactctca acagttcgct taggcatgga agttttgcgg
     7921 cattctggct acacaataac aagggaagac ttactcgtgg ctgcaaccct actagctcaa
     7981 aatttattca cacatggtta cgctttgggg aaattatgag gggatctctc agcgttaagg
     8041 gattttggtc atgcattcta ggtgattaga aaaactcatc gagcatcaaa tgaaactgca
     8101 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     8161 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     8221 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     8281 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     8341 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     8401 ccaaaccgtt attcattcgt gattgcgcct gagcgagtcg aaatacgcga tcgctgttaa
     8461 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     8521 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttccctggga
     8581 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     8641 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     8701 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcggt
     8761 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     8821 catccatgtt ggaatttaat cgcggccttg agcaagacgt ttcccgttga atatggctca
     8881 t
//
