LOCUS       sequence-321964-        8922 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TU for wtGFP expression driven by 2x35s promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8922)
  TITLE     Orzaez lab plasmids
REFERENCE   2  (bases 1 to 8922)
  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 P35Sx2_GFP_Tnos (GB2980)
FEATURES             Location/Qualifiers
     source          1..8922
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1235..1259
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             1692..2507
                     /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             1692..2507
                     /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     complement(1765..1784)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2595..3183
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3084..3103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3337..3354
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3369..3509
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3435..3454)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3595..3617
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      3853..4047
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(4113..5186)
                     /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"
     CDS             complement(5615..6244)
                     /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    7545..7569
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     7757..7775
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(7810..8062)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(join(8086..8799,8800..8802,8803..8805))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(8131..8152)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     8480..8499
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8739..8760
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(join(8879..8922,1..301))
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     complement(join(8915..8922,1..13))
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
ORIGIN
        1 ttgcgaagga tagtgggatt gtgcgtcatc ccttacgtca gtggagatat cacatcaatc
       61 cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg
      121 gggtccatct ttgggaccac tgtcggcagg ggcatcttga acgatagcct ttcctttatc
      181 gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga tgaagtgaca
      241 gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt gaaaagtctc
      301 attgcgaagg atagtgggat tgtgcgtcat cccttacgtc agtggagata tcacatcaat
      361 ccacttgctt tgaagacgtg gttggaacgt cttctttttc cacgatgctc ctcgtgggtg
      421 ggggtccatc tttgggacca ctgtcggcag gggcatcttg aacgatagcc tttcctttat
      481 cgcaatgatg gcatttgtag gtgccacctt ccttttctac tgtccttttg atgaagtgac
      541 agatagctgg gcaatggaat ccgaggaggt ttcccgatat taccctttgt tgaaaagtct
      601 caatagccct ttggtcttct gagactgtat ctttgatatt cttggagtag acgagagtgt
      661 cgtgctccac catgttgacg aagattttct tcttgtcatt gagtcgtaaa agactctgta
      721 tgaactgttc gccagtcttc acggcgagtt ctgttagatc ctcgatctga atttttgact
      781 ccatggcctt tgattcagta ggaactactt tcttagagac tccaatctct attacttgcc
      841 ttggtttatg aagcaagcct tgaatcgtcc atactggaat agtacttctg atcttgagaa
      901 atatatcttt ctctgtgttc ttgatgcagt tagtcctgaa tcttttgact gcatctttaa
      961 ccttcttggg aaggtatttg atctcctgga gattattact cgggtagatc gtcttgatca
     1021 gacctgccgc gtaggcctct ctaaccatct gtgggtcagc attctttctg aaattgaaga
     1081 ggctaatctt ctcattatcg gtggtgaaca tggtatcgtc accttctccg tcgaactttc
     1141 ttcctagatc gtagagatag agaaagtcgt ccatggtgat ctccggggca aaggagatca
     1201 gcttggctct agtctcctga gacgaattcg ttatgtttac accacaatat atcctgccac
     1261 cagccagcca acagctcccc gaccggcagc tcggcacaaa atcaccactc gatacaggca
     1321 gcccatcagt ccgggacggc gtcagcggga gagccgttgt aaggcggcag actttgctca
     1381 tgttaccgat gctattcgga agaacggcaa ctaagctgcc gggtttgaaa cacggatgat
     1441 ctcgcggagg gtagcatgtt gattgtaacg atgacagagc gttgctgcct gtgatcaccg
     1501 cggtttcaaa atcggctccg tcgatactat gttatacgcc aactttgaaa acaactttga
     1561 aaaagctgtt ttctggtatt taaggtttta gaatgcaagg aacagtgaat tggagttcgt
     1621 cttgttataa ttagcttctt ggggtatctt taaatactgt agaaaagagg aaggaaataa
     1681 taagttctgt tatgagccat attcaacggg aaacgtcttg ctcaaggccg cgattaaatt
     1741 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     1801 gtgcgacaat ctaccgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     1861 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     1921 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     1981 tcaccactgc gatcccaggg aaaacagcat tccaggtatt agaagaatat cctgattcag
     2041 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     2101 gtaattgtcc ttttaacagc gatcgcgtat ttcgactcgc tcaggcgcaa tcacgaatga
     2161 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     2221 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     2281 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     2341 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     2401 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     2461 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca cctagaatgc
     2521 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2581 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2641 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2701 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     2761 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2821 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     2881 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     2941 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3001 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3061 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3121 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3181 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3241 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3301 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3361 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3421 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     3481 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     3541 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtcaccggga
     3601 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ggtgccttga
     3661 tgtgggcgcc ggcggtcgag tggcgacggc gcggcttgtc cgcgccctgg tagattgcct
     3721 ggccgtaggc cagccatttt tgagcggcca gcggccgcga taggccgacg cgaagcggcg
     3781 gggcgtaggg agcgcagcga ccgaagggta ggcgcttttt gcagctcttc ggctgtgcgc
     3841 tggccagaca gttatgcaca ggccaggcgg gttttaagag ttttaataag ttttaaagag
     3901 ttttaggcgg aaaaatcgcc ttttttctct tttatatcag tcacttacat gtgtgaccgg
     3961 ttcccaatgt acggctttgg gttcccaatg tacgggttcc ggttcccaat gtacggcttt
     4021 gggttcccaa tgtacgtgct atccacagga aagagtcctt ttcgaccttt ttcccctgct
     4081 agggcaattt gccctagcat ctgctccgta cattaggaac cggcggatgc ttcgccctcg
     4141 atcaggttgc ggtagcgcat gactaggatc gggccagcct gccccgcctc ctccttcaaa
     4201 tcgtactccg gcaggtcatt tgacccgatc agcttgcgca cggtgaaaca gaacttcttg
     4261 aactctccgg cgctgccact gcgttcgtag atcgtcttga acaaccatct ggcttctgcc
     4321 ttgcctgcgg cgcggcgtgc caggcggtag agaaaacggc cgatgccggg atcgatcaaa
     4381 aagtaatcgg ggtgaaccgt cagcacgtcc gggttcttgc cttctgtgat ctcgcggtac
     4441 atccaatcag ctagctcgat ctcgatgtac tccggccgcc cggtttcgct ctttacgatc
     4501 ttgtagcggc taatcaaggc ttcaccctcg gataccgtca ccaggcggcc gttcttggcc
     4561 ttcttcgtac gctgcatggc aacgtgcgtg gtgtttaacc gaatgcaggt ttctaccagg
     4621 tcgtctttct gctttccgcc atcggctcgc cggcagaact tgagtacgtc cgcaacgtgt
     4681 ggacggaaca cgcggccggg cttgtctccc ttcccttccc ggtatcggtt catggattcg
     4741 gttagatggg aaaccgccat cagtaccagg tcgtaatccc acacactggc catgccggcc
     4801 ggccctgcgg aaacctctac gtgcccgtct ggaagctcgt agcggatcac ctcgccagct
     4861 cgtcggtcac gcttcgacag acggaaaacg gccacgtcca tgatgctgcg actatcgcgg
     4921 gtgcccacgt catagagcat cggaacgaaa aaatctggtt gctcgtcgcc cttgggcggc
     4981 ttcctaatcg acggcgcacc ggctgccggc ggttgccggg attctttgcg gattcgatca
     5041 gcggccgctt gccacgattc accggggcgt gcttctgcct cgatgcgttg ccgctgggcg
     5101 gcctgcgcgg ccttcaactt ctccaccagg tcatcaccca gcgccgcgcc gatttgtacc
     5161 gggccggatg gtttgcgacc gctcacgccg attcctcggg cttgggggtt ccagtgccat
     5221 tgcagggccg gcaggcaacc cagccgctta cgcctggcca accgcccgtt cctccacaca
     5281 tggggcattc cacggcgtcg gtgcctggtt gttcttgatt ttccatgccg cctcctttag
     5341 ccgctaaaat tcatctactc atttattcat ttgctcattt actctggtag ctgcgcgatg
     5401 tattcagata gcagctcggt aatggtcttg ccttggcgta ccgcgtacat cttcagcttg
     5461 gtgtgatcct ccgccggcaa ctgaaagttg acccgcttca tggctggcgt gtctgccagg
     5521 ctggccaacg ttgcagcctt gctgctgcgt gcgctcggac ggccggcact tagcgtgttt
     5581 gtgcttttgc tcattttctc tttacctcat taactcaaat gagttttgat ttaatttcag
     5641 cggccagcgc ctggacctcg cgggcagcgt cgccctcggg ttctgattca agaacggttg
     5701 tgccggcggc ggcagtgcct gggtagctca cgcgctgcgt gatacgggac tcaagaatgg
     5761 gcagctcgta cccggccagc gcctcggcaa cctcaccgcc gatgcgcgtg cctttgatcg
     5821 cccgcgacac gacaaaggcc gcttgtagcc ttccatccgt gacctcaatg cgctgcttaa
     5881 ccagctccac caggtcggcg gtggcccata tgtcgtaagg gcttggctgc accggaatca
     5941 gcacgaagtc ggctgccttg atcgcggaca cagccaagtc cgccgcctgg ggcgctccgt
     6001 cgatcactac gaagtcgcgc cggccgatgg ccttcacgtc gcggtcaatc gtcgggcggt
     6061 cgatgccgac aacggttagc ggttgatctt cccgcacggc cgcccaatcg cgggcactgc
     6121 cctggggatc ggaatcgact aacagaacat cggccccggc gagttgcagg gcgcgggcta
     6181 gatgggttgc gatggtcgtc ttgcctgacc cgcctttctg gttaagtaca gcgataacct
     6241 tcatgcgttc cccttgcgta tttgtttatt tactcatcgc atcatatacg cagcgaccgc
     6301 atgacgcaag ctgttttact caaatacaca tcaccttttt agacggcggc gctcggtttc
     6361 ttcagcggcc aagctggccg gccaggccgc cagcttggca tcagacaaac cggccaggat
     6421 ttcatgcagc cgcacggttg agaggtgcgc gggcggctcg aacacgtacc cggccgcgat
     6481 catctccgcc tcgatctctt cggtaatgaa aaacggttcg tcctggccgt cctggtgcgg
     6541 tttcatgctt gttcctcttg gcgttcattc tcggcggccg ccagggcgtc ggcctcggtc
     6601 aatgcgtcct cacggaaggc accgcgccgc ctggcctcgg tgggcgtcac ttcctcgctg
     6661 cgctcaagtg cgcggtacag ggtcgagcga tgcacgccaa gcagtgcagc cgcctctttc
     6721 acggtgcggc cttcctggtc gatcagctcg cgggcgtgcg cgatctgtgc cggggtgagg
     6781 gtagggcggg ggccaaactt cacgcctcgg gccttggcgg cctcgcgccc gctccgggtg
     6841 cggtcgatga ttagggaacg ctcgaactcg gcaatgccgg cgaacacggt caacaccatg
     6901 cggccggccg gcgtggtggt gtcggcccac ggctctgcca ggctacgcag gcccgcgccg
     6961 gcctcctgga tgcgctcggc aatgtccagt aggtcgcggg tgctgcgggc caggcggtct
     7021 agcctggtca ctgtcacaac gtcgccaggg cgtaggtggt caagcatcct ggccagctcc
     7081 gggcggtcgc gcctggtgcc ggtgatcttc tcggaaaaca gcttggtgca gccggccgcg
     7141 tgcagttcgg cccgttggtt ggtcaagtcc tggtcgtcgg tgctgacgcg ggcatagccc
     7201 agcaggccag cggcggcgct cttgttcatg gcgtaatgtc tccggttcta gtcgcaagta
     7261 ttctacttta tgcgactaaa acacgcgaca agaaaacgcc aggaaaaggg cagggcggca
     7321 gcctgtcgcg taacttagga cttgtgcgac atgtcgtttt cagatgacgg ctgcactgaa
     7381 cgtcagaagc cgactgcact atagcagcgg aggggttgga tcaaagtact ttgatcccga
     7441 ggggaaccct gtggttggca tgcacataca aatggacgaa cggataaacc ttttcacgcc
     7501 cttttaaata tccgattatt ctaataaacg ctcttttctc ttaggtttac ccgccaatat
     7561 atcctgtcag aattcgtctc atgacagcgc gagtcggtcc cattattgaa gcatttatca
     7621 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7681 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     7741 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcaag aattagcttg
     7801 gcaattcccg atctagtaac atagatgaca ccgcgcgcga taatttatcc tagtttgcgc
     7861 gctatatttt gttttctatc gcgtattaaa tgtataattg cgggactcta atcataaaaa
     7921 cccatctcat aaataacgtc atgcattaca tgttaattat tacatgctta acgtaattca
     7981 acagaaatta tatgataatc gtcgcaagac cggcaacagg attcaatctt aagaaacttt
     8041 attgccaaat gtttgaacga tcgggatcga agatccattc caagctcact tgtacagctc
     8101 gtccatgccg tgagtgatcc cggcggcggt cacgaactcc agcaggacca tgtgatcgcg
     8161 cttctcgttg gggtctttgc tcagggcgga ctgggtgctc aggtagtggt tgtcgggcag
     8221 cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga gctgcacgct
     8281 gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg atgccgttct tctgcttgtc
     8341 ggccatgata tagacgttgt ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt
     8401 gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc
     8461 gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc
     8521 ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca tgtggtcggg
     8581 gtagcggctg aagcactgca cgccgtagct gaaggtggtc acgagggtgg gccagggcac
     8641 gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg tggcatcgcc
     8701 ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag
     8761 gatgggcacc accccggtga acagctcctc gcccttgctc accatttcga ctagaatagt
     8821 aaattgtaat gttgtttgtt gtttgttttg ttgtggtaat tgttgtaaaa ataccggagt
     8881 cctctccaaa tgaaatgaac ttccttatat agaggaaggg tc
//
