LOCUS       sequence-368896-        7220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eGFP under the virB promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7220)
  AUTHORS   Aliu E, Lee K, Wang K
  TITLE     CRISPR RNA-guided integrase enables high-efficiency targeted genome 
            engineering in Agrobacterium tumefaciens.
  JOURNAL   Plant Biotechnol J. 2022 Jun 11. doi: 10.1111/pbi.13872.
  PUBMED    35690588
REFERENCE   2  (bases 1 to 7220)
  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: "Plant 
            Biotechnol J. 2022 Jun 11. doi: 10.1111/pbi.13872."
COMMENT     Sequence Label: pEA183
FEATURES             Location/Qualifiers
     source          1..7220
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..195
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(431..453)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    539..679
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     594..613
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(694..711)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(865..1453)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(945..964)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1699..2493)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MAREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKP
                     HSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPA
                     KRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDL
                     FEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQ
                     PVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     RBS             2922..2933
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(2940..2942,2943..2945,2946..3659)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2940..2942,2943..2945,2946..3656)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2985..3006)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3246..3265)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3593..3614
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      3675..3746
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(3747..3774)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             5024..5653
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             6082..7155
                     /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"
ORIGIN
        1 tgtggatagc acgtacattg ggaacccaaa gccgtacatt gggaaccgga acccgtacat
       61 tgggaaccca aagccgtaca ttgggaaccg gtcacacatg taagtgactg atataaaaga
      121 gaaaaaaggc gatttttccg cctaaaactc tttaaaactt attaaaactc ttaaaacccg
      181 cctggcctgt gcataactgt ctggccagcg cacagccgaa gagctgcaaa aagcgcctac
      241 ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg
      301 ccgctcaaaa atggctggcc tacggccagg caatctacca gggcgcggac aagccgcgcc
      361 gtcgccactc gaccgccggc gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg
      421 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
      481 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
      541 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
      601 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
      661 gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
      721 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
      781 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
      841 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
      901 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
      961 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     1021 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     1081 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     1141 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     1201 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     1261 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     1321 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     1381 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     1441 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     1501 aaactcacgt taagggattt tggtcatgca tgatatatct cccaatttgt gtagggctta
     1561 ttatgcacgc ttaaaaataa taaaagcaga cttgacctga tagtttggct gtgagcaatt
     1621 atgtgcttag tgcatctaac gcttgagtta agccgcgccg cgaagcggcg tcggcttgaa
     1681 cgaatttcta gctagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg
     1741 gacaaattct tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata
     1801 agcctgtcta gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca
     1861 gtcggcagcg acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga
     1921 caacgtaagc actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt
     1981 taaggtttca tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc
     2041 cgccgctgga cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag
     2101 atcaatgtcg attgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc
     2161 tccaaattgc agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac
     2221 aatggtgact tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa
     2281 aaggtcgttg atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag
     2341 caaatcaata tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac
     2401 ggccagcaac gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt
     2461 tgatacttcg gcgataaccg cttcacgagc catggatccc tccctacaac tagtgatatc
     2521 gcccggagtt cgctcccaga aatatagcca tccctgcccc ctcacgtcaa tcccacgagc
     2581 ctcttatcca ttctcattga acgacggcga attgtattga ttggtgagct aatcaatatc
     2641 aaatatgaaa gggaaatcgt taaattattc aattattatc taattgaata atttaagaat
     2701 aatggtacct ccggttgctt agtcatggat gcgcggttct cggtccatgt tgcgttccaa
     2761 gacgccgggc gaggtttctc gcttcaattg aaatcataaa gaagcaattg aaaattttcg
     2821 agtaaccgac cctcccgata atcttcaaca taaaacaacg cacttcttcc aacgggagag
     2881 gcggtgttag ttgcgagcta aggagataag gtgaattcat taaagaggag aaaacgcgta
     2941 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     3001 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     3061 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     3121 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     3181 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     3241 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     3301 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     3361 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     3421 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     3481 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     3541 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     3601 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa
     3661 agctttaagg atctcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     3721 tctgttgttt gtcggtgaac gctctccgca gaaaggccca cccgaaggtg agccgcatgc
     3781 caaccacagg gttcccctcg ggatcaaagt actttaaagt actttaaagt actttaaagt
     3841 actttgatcc aacccctccg ctgctatagt gcagtcggct tctgacgttc agtgcagccg
     3901 tcttctgaaa acgacatgtc gcacaagtcc taagttacgc gacaggctgc cgccctgccc
     3961 ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa agtagaatac ttgcgactag
     4021 aaccggagac attacgccat gaacaagagc gccgccgctg gcctgctggg ctatgcccgc
     4081 gtcagcaccg acgaccagga cttgaccaac caacgggccg aactgcacgc ggccggctgc
     4141 accaagctgt tttccgagaa gatcaccggc accaggcgcg accgcccgga gctggccagg
     4201 atgcttgacc acctacgccc tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc
     4261 cgcagcaccc gcgacctact ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg
     4321 cgtagcctgg cagagccgtg ggccgacacc accacgccgg ccggccgcat ggtgttgacc
     4381 gtgttcgccg gcattgccga gttcgagcgt tccctaatca tcgaccgcac ccggagcggg
     4441 cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc gccctaccct caccccggca
     4501 cagatcgcgc acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa agaggcggct
     4561 gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg
     4621 acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac
     4681 gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg caccaggacg
     4741 gccaggacga accgtttttc attaccgaag agatcgaggc ggagatgatc gcggccgggt
     4801 acgtgttcga gccgcccgcg cacgtctcaa ccgtgcggct gcatgaaatc ctggccggtt
     4861 tgtctgatgc caagctggcg gcctggccgg ccagcttggc cgctgaagaa accgagcgcc
     4921 gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt
     4981 atatgatgcg atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt
     5041 acttaaccag aaaggcgggt caggcaagac gaccatcgca acccatctag cccgcgccct
     5101 gcaactcgcc ggggccgatg ttctgttagt cgattccgat ccccagggca gtgcccgcga
     5161 ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat
     5221 tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg gagcgcccca
     5281 ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca
     5341 gccaagccct tacgacatat gggccaccgc cgacctggtg gagctggtta agcagcgcat
     5401 tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac
     5461 gcgcatcggc ggtgaggttg ccgaggcgct ggccgggtac gagctgccca ttcttgagtc
     5521 ccgtatcacg cagcgcgtga gctacccagg cactgccgcc gccggcacaa ccgttcttga
     5581 atcagaaccc gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc
     5641 aaaactcatt tgagttaatg aggtaaagag aaaatgagca aaagcacaaa cacgctaagt
     5701 gccggccgtc cgagcgcacg cagcagcaag gctgcaacgt tggccagcct ggcagacacg
     5761 ccagccatga agcgggtcaa ctttcagttg ccggcggagg atcacaccaa gctgaagatg
     5821 tacgcggtac gccaaggcaa gaccattacc gagctgctat ctgaatacat cgcgcagcta
     5881 ccagagtaaa tgagcaaatg aataaatgag tagatgaatt ttagcggcta aaggaggcgg
     5941 catggaaaat caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac
     6001 gggcggttgg ccaggcgtaa gcggctgggt tgtctgccgg ccctgcaatg gcactggaac
     6061 ccccaagccc gaggaatcgg cgtgagcggt cgcaaaccat ccggcccggt acaaatcggc
     6121 gcggcgctgg gtgatgacct ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa
     6181 cgcatcgagg cagaagcacg ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc
     6241 aaagaatccc ggcaaccgcc ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc
     6301 gacgagcaac cagatttttt cgttccgatg ctctatgacg tgggcacccg cgatagtcgc
     6361 agcatcatgg acgtggccgt tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg
     6421 atccgctacg agcttccaga cgggcacgta gaggtttccg cagggccggc cggcatggcc
     6481 agtgtgtggg attacgacct ggtactgatg gcggtttccc atctaaccga atccatgaac
     6541 cgataccggg aagggaaggg agacaagccc ggccgcgtgt tccgtccaca cgttgcggac
     6601 gtactcaagt tctgccggcg agccgatggc ggaaagcaga aagacgacct ggtagaaacc
     6661 tgcattcggt taaacaccac gcacgttgcc atgcagcgta cgaagaaggc caagaacggc
     6721 cgcctggtga cggtatccga gggtgaagcc ttgattagcc gctacaagat cgtaaagagc
     6781 gaaaccgggc ggccggagta catcgagatc gagctagctg attggatgta ccgcgagatc
     6841 acagaaggca agaacccgga cgtgctgacg gttcaccccg attacttttt gatcgatccc
     6901 ggcatcggcc gttttctcta ccgcctggca cgccgcgccg caggcaaggc agaagccaga
     6961 tggttgttca agacgatcta cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt
     7021 ttcaccgtgc gcaagctgat cgggtcaaat gacctgccgg agtacgattt gaaggaggag
     7081 gcggggcagg ctggcccgat cctagtcatg cgctaccgca acctgatcga gggcgaagca
     7141 tccgccggtt cctaatgtac ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa
     7201 ggtcgaaaag gtctctttcc
//
