LOCUS       sequence-321967-        7313 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multiplex construct with 2.1 aptamer in scf to target -104 -162 -229
            positions of 35s promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7313)
  TITLE     Orzaez lab plasmids
REFERENCE   2  (bases 1 to 7313)
  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"
COMMENT     Sequence Label: pEGB3_alpha1 gRNAs 2.1 P35S -104 -162 -229 (GB...
FEATURES             Location/Qualifiers
     source          1..7313
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        308..383
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        538..613
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        770..845
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(912..936)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    962..986
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             2287..2916
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             3345..4418
                     /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      4484..4678
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(4914..4936)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5022..5162
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5077..5096
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5177..5194)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5348..5936)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5428..5447)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6024..6839)
                     /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(6024..6839)
                     /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     6747..6766
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    7272..7296
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 ggagcatctt cattcttaag atatgaagat aatcttcaaa aggcccctgg gaatctgaaa
       61 gaagagaagc aggcccattt atatgggaaa gaacaatagt atttcttata taggcccatt
      121 taagttgaaa acaatcttca aaagtcccac atcgcttaga taagaaaacg aagctgagtt
      181 tatatacagc tagagtcgaa gtagtgattg aacaaagcac cagtggtcta gtggtagaat
      241 agtaccctgc cacggtacag acccgggttc gattcccggc tggtgcaaac cacgtcttca
      301 aagcaaggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
      361 aaaaagtggc accgagtcgg tgcgggagca catgaggaat cacccatgtg cgactcccac
      421 agtcactggg gagtcttccc aacaaagcac cagtggtcta gtggtagaat agtaccctgc
      481 cacggtacag acccgggttc gattcccggc tggtgcaacg atgctcctcg tgggtgggtt
      541 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      601 accgagtcgg tgcgggagca catgaggaat cacccatgtg cgactcccac agtcactggg
      661 gagtcttccc aaaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac
      721 agacccgggt tcgattcccg gctggtgcac ctttccttta tcgcaatgag ttttagagct
      781 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
      841 ggtgcgggag cacatgagga atcacccatg tgcgactccc acagtcactg gggagtcttc
      901 ccttttttct gacccagctt tcttgtacaa agttggcatt acgctgtcat gagacgaatt
      961 ctgacaggat atattggcgg gtaaacctaa gagaaaagag cgtttattag aataatcgga
     1021 tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac
     1081 agggttcccc tcgggatcaa agtactttga tccaacccct ccgctgctat agtgcagtcg
     1141 gcttctgacg ttcagtgcag ccgtcatctg aaaacgacat gtcgcacaag tcctaagtta
     1201 cgcgacaggc tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca
     1261 taaagtagaa tacttgcgac tagaaccgga gacattacgc catgaacaag agcgccgccg
     1321 ctggcctgct gggctatgcc cgcgtcagca ccgacgacca ggacttgacc aaccaacggg
     1381 ccgaactgca cgcggccggc tgcaccaagc tgttttccga gaagatcacc ggcaccaggc
     1441 gcgaccgccc ggagctggcc aggatgcttg accacctacg ccctggcgac gttgtgacag
     1501 tgaccaggct agaccgcctg gcccgcagca cccgcgacct actggacatt gccgagcgca
     1561 tccaggaggc cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac accaccacgc
     1621 cggccggccg catggtgttg accgtgttcg ccggcattgc cgagttcgag cgttccctaa
     1681 tcatcgaccg cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc
     1741 cccgccctac cctcaccccg gcacagatcg cgcacgcccg cgagctgatc gaccaggaag
     1801 gccgcaccgt gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg
     1861 cacttgagcg cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt gccttccgtg
     1921 aggacgcatt gaccgaggcc gacgccctgg cggccgccga gaatgaacgc caagaggaac
     1981 aagcatgaaa ccgcaccagg acggccagga cgaaccgttt ttcattaccg aagagatcga
     2041 ggcggagatg atcgcggccg ggtacgtgtt cgagccgccc gcgcacctct caaccgtgcg
     2101 gctgcatgaa atcctggccg gtttgtctga tgccaagctg gcggcctggc cggccagctt
     2161 ggccgctgaa gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag
     2221 cttgcgtcat gcggtcgctg cgtatatgat gcgatgagta aataaacaaa tacgcaaggg
     2281 gaacgcatga aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc
     2341 gcaacccatc tagcccgcgc cctgcaactc gccggggccg atgttctgtt agtcgattcc
     2401 gatccccagg gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt
     2461 gtcggcatcg accgcccgac gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc
     2521 gtagtgatcg acggagcgcc ccaggcggcg gacttggctg tgtccgcgat caaggcagcc
     2581 gacttcgtgc tgattccggt gcagccaagc ccttacgaca tatgggccac cgccgacctg
     2641 gtggagctgg ttaagcagcg cattgaggtc acggatggaa ggctacaagc ggcctttgtc
     2701 gtgtcgcggg cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg
     2761 tacgagctgc ccattcttga gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc
     2821 gccgccggca caaccgttct tgaatcagaa cccgagggcg acgctgcccg cgaggtccag
     2881 gcgctggccg ctgaaattaa atcaaaactc atttgagtta atgaggtaaa gagaaaatga
     2941 gcaaaagcac aaacacgcta agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa
     3001 cgttggccag cctggcagac acgccagcca tgaagcgggt caactttcag ttgccggcgg
     3061 aggatcacac caagctgaag atgtacgcgg tacgccaagg caagaccatt accgagctgc
     3121 tatctgaata catcgcgcag ctaccagagt aaatgagcaa atgaataaat gagtagatga
     3181 attttagcgg ctaaaggagg cggcatggaa aatcaagaac aaccaggcac cgacgccgtg
     3241 gaatgcccca tgtgtggagg aacgggcggt tggccaggcg taagcggctg ggttgcctgc
     3301 cggccctgca atggcactgg aacccccaag cccgaggaat cggcgtgagc ggtcgcaaac
     3361 catccggccc ggtacaaatc ggcgcggcgc tgggtgatga cctggtggag aagttgaagg
     3421 ccgcgcaggc cgcccagcgg caacgcatcg aggcagaagc acgccccggt gaatcgtggc
     3481 aagcggccgc tgatcgaatc cgcaaagaat cccggcaacc gccggcagcc ggtgcgccgt
     3541 cgattaggaa gccgcccaag ggcgacgagc aaccagattt tttcgttccg atgctctatg
     3601 acgtgggcac ccgcgatagt cgcagcatca tggacgtggc cgttttccgt ctgtcgaagc
     3661 gtgaccgacg agctggcgag gtgatccgct acgagcttcc agacgggcac gtagaggttt
     3721 ccgcagggcc ggccggcatg gccagtgtgt gggattacga cctggtactg atggcggttt
     3781 cccatctaac cgaatccatg aaccgatacc gggaagggaa gggagacaag cccggccgcg
     3841 tgttccgtcc acacgttgcg gacgtactca agttctgccg gcgagccgat ggcggaaagc
     3901 agaaagacga cctggtagaa acctgcattc ggttaaacac cacgcacgtt gccatgcagc
     3961 gtacgaagaa ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa gccttgatta
     4021 gccgctacaa gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag atcgagctag
     4081 ctgattggat gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg acggttcacc
     4141 ccgattactt tttgatcgat cccggcatcg gccgttttct ctaccgcctg gcacgccgcg
     4201 ccgcaggcaa ggcagaagcc agatggttgt tcaagacgat ctacgaacgc agtggcagcg
     4261 ccggagagtt caagaagttc tgtttcaccg tgcgcaagct gatcgggtca aatgacctgc
     4321 cggagtacga tttgaaggag gaggcggggc aggctggccc gatcctagtc atgcgctacc
     4381 gcaacctgat cgagggcgaa gcatccgccg gttcctaatg tacggagcag atgctagggc
     4441 aaattgccct agcaggggaa aaaggtcgaa aaggactctt tcctgtggat agcacgtaca
     4501 ttgggaaccc aaagccgtac attgggaacc ggaacccgta cattgggaac ccaaagccgt
     4561 acattgggaa ccggtcacac atgtaagtga ctgatataaa agagaaaaaa ggcgattttt
     4621 ccgcctaaaa ctctttaaaa cttattaaaa ctcttaaaac ccgcctggcc tgtgcataac
     4681 tgtctggcca gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg tcgctgcgct
     4741 ccctacgccc cgccgcttcg cgtcggccta tcgcggccgc tggccgctca aaaatggctg
     4801 gcctacggcc aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca ctcgaccgcc
     4861 ggcgcccaca tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     4921 cacatgcagc tcccggtgac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     4981 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     5041 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga
     5101 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     5161 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5221 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5281 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5341 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5401 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5461 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5521 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5581 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5641 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5701 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5761 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     5821 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5881 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5941 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6001 ttttggtcat gcattctagg tgattagaaa aactcatcga gcatcaaatg aaactgcaat
     6061 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     6121 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     6181 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     6241 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     6301 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     6361 aaaccgttat tcattcgtga ttgcgcctga gcgagtcgaa atacgcgatc gctgttaaaa
     6421 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     6481 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt ccctgggatc
     6541 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     6601 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     6661 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcggtag
     6721 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     6781 tccatgttgg aatttaatcg cggccttgag caagacgttt cccgttgaat atggctcata
     6841 acagaactta ttatttcctt cctcttttct acagtattta aagatacccc aagaagctaa
     6901 ttataacaag acgaactcca attcactgtt ccttgcattc taaaacctta aataccagaa
     6961 aacagctttt tcaaagttgt tttcaaagtt ggcgtataac atagtatcga cggagccgat
     7021 tttgaaaccg cggtgatcac aggcagcaac gctctgtcat cgttacaatc aacatgctac
     7081 cctccgcgag atcatccgtg tttcaaaccc ggcagcttag ttgccgttct tccgaatagc
     7141 atcggtaaca tgagcaaagt ctgccgcctt acaacggctc tcccgctgac gccgtcccgg
     7201 actgatgggc tgcctgtatc gagtggtgat tttgtgccga gctgccggtc ggggagctgt
     7261 tggctggctg gtggcaggat atattgtggt gtaaacataa cgaattcgtc tca
//
