LOCUS       sequence-213500-        5510 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5510)
  AUTHORS   Stuurman N, Pacios Bras C, Schlaman HR, Wijfjes AH, Bloemberg G, 
            Spaink HP
  TITLE     Use of green fluorescent protein color variants expressed on stable 
            broad-host-range vectors to visualize rhizobia interacting with 
            plants.
  JOURNAL   Mol Plant Microbe Interact. 2000 Nov;13(11):1163-9. doi: 
            10.1094/MPMI.2000.13.11.1163.
  PUBMED    11059482
REFERENCE   2  (bases 1 to 5510)
  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"; doi: 
            "10.1094/MPMI.2000.13.11"; journalName: "Mol Plant Microbe 
            Interact"; date: "2000-11"; volume: "13"; issue: "11"; pages: 
            "1163-9"
COMMENT     Sequence Label: pMP2463
FEATURES             Location/Qualifiers
     source          1..5510
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..27
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     24..40
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..40
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     59..79
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        61..79
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     108..124
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     109..125
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      171..180
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(177..179,180..182,183..896)
                     /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(177..179,180..182,183..893)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(222..243)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(483..502)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     830..851
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(946..965)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(947..965)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(975..992)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(975..991)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(984..1006)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(1700..2362)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      2363..3134
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             complement(4416..4949)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(5138..5143,5144..5160,5161..5166))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    5436..5457
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5472..5477,5478..5495,5496..5502)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(5510,1..16)
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
ORIGIN
        1 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc
       61 aattaaccct cactaaaggg aacaaaagct gggtaccggg ccccccctcg aggtcgacgg
      121 tatcgataag cttgatatcg aattcctgca gcccggggga tccaccggtc gccaccatgg
      181 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
      241 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
      301 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
      361 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
      421 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
      481 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
      541 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
      601 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
      661 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
      721 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
      781 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
      841 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaagcg
      901 gccgcgactc tagagcggcc gccaccgcgg tggagctcca attcgcccta tagtgagtcg
      961 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     1021 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     1081 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gaaattgtaa
     1141 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     1201 aataggccgt actgcgatga gtggcagggc ggggcgtaat ttttttaagg cagttattgg
     1261 tgcccttaaa cgcctggttg ctacgcctga ataagtgata ataagcggat gaatggcaga
     1321 aattcgaaag caaattcgac ccggtcgtcg gttcagggca gggtcgttaa atagccgctt
     1381 atgtctattg ctggtttacc ggtttattga ctaccggaag cagtgtgacc gtgtgcttct
     1441 caaatgcctg aggccagttt gctcaggctc tccccgtgga ggtaataatt gacgatatga
     1501 tcatttattc tgcctcccag agcctgataa aaacggtgaa tccgttagcg aggtgccgcc
     1561 ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg cggggaggca
     1621 gacaaggtat agggcggcga ggcggctaca gccgatagtc tggaacagcg cacttacggg
     1681 ttgctgcgca acccaagtgc taccggcgcg gcagcgtgac ccgtgtcggc ggctccaacg
     1741 gctcgccatc gtccagaaaa cacggctcat cgggcatcgg caggcgctgc tgcccgcgcc
     1801 gttcccattc ctccgtttcg gtcaaggctg gcaggtctgg ttccatgccc ggaatgccgg
     1861 gctggctggg cggctcctcg ccggggccgg tcggtagttg ctgctcgccc ggatacaggg
     1921 tcgggatgcg gcgcaggtcg ccatgcccca acagcgattc gtcctggtcg tcgtgatcaa
     1981 ccaccacggc ggcactgaac accgacaggc gcaactggtc gcggggctgg ccccacgcca
     2041 cgcggtcatt gaccacgtag gccgacacgg tgccggggcc gttgagcttc acgacggaga
     2101 tccagcgctc ggccaccaag tccttgactg cgtattggac cgtccgcaaa gaacgtccga
     2161 tgagcttgga aagtgtcttc tggctgacca ccacggcgtt ctggtggccc atctgcgcca
     2221 cgaggtgatg cagcagcatt gccgccgtgg gtttcctcgc aataagcccg gcccacgcct
     2281 catgcgcttt gcgttccgtt tgcacccagt gaccgggctt gttcttggct tgaatgccga
     2341 tttctctgga ctgcgtggcc atgcttatct ccatgcggta ggggtgccgc acggttgcgg
     2401 caccatgcgc aatcagctgc aacttttcgg cagcgcgaca acaattatgc gttgcgtaaa
     2461 agtggcagtc aattacagat tttctttaac ctacgcaatg agctattgcg gggggtgccg
     2521 caatgagctg ttgcgtaccc ccctttttta agttgttgat ttttaagtct ttcgcatttc
     2581 gccctatatc tagttctttg gtgcccaaag aagggcaccc ctgcggggtt cccccacgcc
     2641 ttcggcgcgg ctccccctcc ggcaaaaagt ggcccctccg gggcttgttg atcgactgcg
     2701 cggccttcgg ccttgcccaa ggtggcgctg cccccttgga acccccgcac tcgccgccgt
     2761 gaggctcggg gggcaggcgg gcgggcttcg cccttcgact gcccccactc gcataggctt
     2821 gggtcgttcc aggcgcgtca aggccaagcc gctgcgcggt cgctgcgcga gccttgaccc
     2881 gccttccact tggtgtccaa ccggcaagcg aagcgcgcag gccgcaggcc ggaggctttt
     2941 ccccagagaa aattaaaaaa attgatgggg caaggccgca ggccgcgcag ttggagccgg
     3001 tgggtatgtg gtcgaaggct gggtagccgg tgggcaatcc ctgtggtcaa gctcgtgggc
     3061 aggcgcagcc tgtccatcag cttgtccagc agggttgtcc acgggccgag cgaagcgagc
     3121 cagccggtgg ccgctcgcgg ccatcgtcca catatccacg ggctggcaag ggagcgcagc
     3181 gaccgcgcag ggcgaagccc ggagagcaag cccgtagggc gccgcagccg ccgtaggcgg
     3241 tcacgacttt gcgaagcaaa gtctagtgag tatactcaag cattgagtgg cccgccggag
     3301 gcaccgcctt gcgctgcccc cgtcgagccg gttggacacc aaaagggagg ggcaggcatg
     3361 gcggcatacg cgatcatgcg atgcaagaag ctggcgaaaa tgggcaacgt ggcggccagt
     3421 ctcaagcacg cctaccgcga gcgcgagacg cccaacgctg acgccagcag gacgccagag
     3481 aacgagcact gggcggccag cagcaccgat gaagcgatgg gccgactgcg cgagttgctg
     3541 ccagagaagc ggcgcaagga cgctgtgttg gcggtcgagt acgtcatgac ggccagcccg
     3601 gaatggtgga agtcggccag ccaagaacag caggcggcgt tcttcgagaa ggcgcacaag
     3661 tggctggcgg acaagtacgg ggcggatcgc atcgtgacgg ccagcatcca ccgtgacgaa
     3721 accagcccgc acatgaccgc gttcgtggtg ccgctgacgc aggacggcag gctgtcggcc
     3781 aaggagttca tcggcaacaa agcgcagatg acccgcgacc agaccacgtt tgcggccgct
     3841 gtggccgatc tagggctgca acggggcatc gagggcagca aggcacgtca cacgcgcatt
     3901 caggcgttct acgaggccct ggagcggcca ccagtgggcc acgtcaccat cagcccgcaa
     3961 gcggtcgagc cacgcgccta tgcaccgcag ggattggccg aaaagctggg aatctcaaag
     4021 cgcgttgaga cgccggaagc cgtggccgac cggctgacaa aagcggttcg gcaggggtat
     4081 gagcctgccc tacaggccgc cgcaggagcg cgtgagatgc gcaagaaggc cgatcaagcc
     4141 caagagacgg cccgagacct tcgggagcgc ctgaagcccg ttctggacgc cctggggccg
     4201 ttgaatcggg atatgcaggc caaggccgcc gcgatcatca aggccgtggg cgaaaagctg
     4261 ctgacggaac agcgggaagt ccagcgccag aaacaggccc agcgccagca ggaacgcggg
     4321 cgcgcacatt tccccgaaaa gtgccacctg cggcgttgtg acaatttacc gaacaactcc
     4381 gcggccggga agccgatctc ggcttgaacg aattgttagg tggcggtact tgggtcgata
     4441 tcaaagtgca tcacttcttc ccgtatgccc aactttgtat agagagccac tgcgggatcg
     4501 tcaccgtaat ctgcttgcac gtagatcaca taagcaccaa gcgcgttggc ctcatgcttg
     4561 aggagattga tgagcgcggt ggcaatgccc tgcctccggt gctctccgga gactgcgaga
     4621 tcatagatat agatctcact acgcggctgc tcaaacttgg gcagaacgta agccgcgaga
     4681 gcgccaacaa ccgcttcttg gtcgaaggca gcaagcgcga tgaatgtctt actacggagc
     4741 aagttcccga ggtaatcgga gtccggctga tgttgggagt aggtggctac gtctccgaac
     4801 tcacgaccga aaagatcaag agcagcccgc atggatttga cttggtcagg gccgagccta
     4861 catgtgcgaa tgatgcccat acttgagcca cctaactttg ttttagggcg actgccctgc
     4921 tgcgtaacat cgttgctgct gcgtaacatc gttgctgctc cataacatca aacatcgacc
     4981 cacggcgtaa cgcgcttgct gcttggatgc ccgaggcata gactgtacaa aaaaacagtc
     5041 ataacaagcc atgaaaaccg ccactgcgcc gttaccaccg ctgcgttcgg tcaaggttct
     5101 ggaccagttg cgtgagcgca tacgctactt gcattacagt ttacgaaccg aacaggctta
     5161 tgtcaactgg gttcgtgcct tcatccgttt ccacggtgtg cgtccatggg caaatattat
     5221 acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat
     5281 ggcttccatg tcggcagaat gcttaatgaa ttacaacagt ttttatgcat gcgcccaata
     5341 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     5401 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     5461 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
//
