LOCUS       sequence-311835-        7312 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 7312)
  TITLE     Orzaez lab plasmids
REFERENCE   2  (bases 1 to 7312)
  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..7312
                     /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="MKVIAVLNQKGGSGKTTIGTHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             3350..4417
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
                     AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
                     DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
                     VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
                     ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
                     RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
                     MRYRNLIEGEASAGS"
     rep_origin      4483..4677
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(4913..4935)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5021..5161
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5076..5095
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5176..5193)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5347..5935)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5427..5446)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6023..6838)
                     /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(6023..6838)
                     /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     6746..6765
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    7271..7295
                     /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 ccgatgagta aataaacaaa tacgcaaggg
     2281 gaacgcatga aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc
     2341 ggaacccatc 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 gatcgcgcag ctaccagagt aaatgagcaa atgaataaat gagtagatga
     3181 attttagcgg ctaaaggagg cggcatggaa aatcaagaac aaccaggcac cgacgccgtg
     3241 gaatgcccca tgtgtggagg aacgggcggt tggccaggcg taagcggctg ggttgtctgc
     3301 cggccctgca atggcactgg aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc
     3361 atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc
     3421 cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca
     3481 agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc
     3541 gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga
     3601 cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg
     3661 tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc
     3721 cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc
     3781 ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt
     3841 gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca
     3901 gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg
     3961 tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag
     4021 ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc
     4081 tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc
     4141 cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc
     4201 cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc
     4261 cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc
     4321 ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg
     4381 caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca
     4441 aattgcccta gcaggggaaa aaggtcgaaa aggactcttt cctgtggata gcacgtacat
     4501 tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta
     4561 cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc
     4621 cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact
     4681 gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc
     4741 cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg
     4801 cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg
     4861 gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     4921 acatgcagct cccggtgacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     4981 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     5041 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     5101 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     5161 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5221 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5281 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5341 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5401 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5461 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5521 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5581 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5641 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5701 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5761 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     5821 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5881 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5941 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6001 tttggtcatg cattctaggt gattagaaaa actcatcgag catcaaatga aactgcaatt
     6061 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     6121 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     6181 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     6241 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     6301 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     6361 aaccgttatt cattcgtgat tgcgcctgag cgagtcgaaa tacgcgatcg ctgttaaaag
     6421 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     6481 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc cctgggatcg
     6541 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     6601 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     6661 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcggtaga
     6721 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     6781 ccatgttgga atttaatcgc ggccttgagc aagacgtttc ccgttgaata tggctcataa
     6841 cagaacttat tatttccttc ctcttttcta cagtatttaa agatacccca agaagctaat
     6901 tataacaaga cgaactccaa ttcactgttc cttgcattct aaaaccttaa ataccagaaa
     6961 acagcttttt caaagttgtt ttcaaagttg gcgtataaca tagtatcgac ggagccgatt
     7021 ttgaaaccgc ggtgatcaca ggcagcaacg ctctgtcatc gttacaatca acatgctacc
     7081 ctccgcgaga tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt ccgaatagca
     7141 tcggtaacat gagcaaagtc tgccgcctta caacggctct cccgctgacg ccgtcccgga
     7201 ctgatgggct gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt
     7261 ggctggctgg tggcaggata tattgtggtg taaacataac gaattcgtct ca
//
