LOCUS       sequence-422938-        3683 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Medium copy number protein expression plasmid, suitable for 
            electroporation in C. necator. Containes eGFP as model protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3683)
  AUTHORS   Arhar S, Rauter T, Stolterfoht-Stock H, Lambauer V, Kratzer R, 
            Winkler M, Karava M, Kourist R, Emmerstorfer-Augustin A
  TITLE     CO(2)-based production of phytase from highly stable expression 
            plasmids in Cupriavidus necator H16.
  JOURNAL   Microb Cell Fact. 2024 Jan 3;23(1):9. doi: 
            10.1186/s12934-023-02280-2.
  PUBMED    38172920
REFERENCE   2  (bases 1 to 3683)
  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: "Microb Cell
            Fact."; date: "2024-01-3"; volume: "23(1):9. doi"; pages: " 
            10.1186/s12934-023-02280-2"
COMMENT     Sequence Label: pKERep
FEATURES             Location/Qualifiers
     source          1..3683
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     279..300
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(384..401)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      554..640
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      732..759
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(857..876)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             868..1683
                     /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             868..1683
                     /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(941..960)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1748..2519
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             2520..3182
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     promoter        join(3195..3209,3210..3215,3216..3232,3233..3239)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3215..3231
                     /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)."
     RBS             3279..3301
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(3309..3311,3312..3314,3315..3683,1..345)
                     /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(3309..3311,3312..3314,3315..3683,1..342)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3354..3375)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3615..3634)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
       61 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
      121 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
      181 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
      241 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
      301 accgccgccg ggatcactct cggcatggac gagctgtaca agtaaaagct tggctgtttt
      361 ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg
      421 ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac
      481 tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg
      541 aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
      601 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa
      661 cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca
      721 tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctttt
      781 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaataataaa actgtctgct
      841 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct
      901 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
      961 aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag
     1021 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     1081 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     1141 gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa
     1201 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     1261 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     1321 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     1381 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     1441 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     1501 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     1561 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     1621 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     1681 taagaattaa ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     1741 cactagtgcg gccaccggct ggctcgcttc gctcggcccg tggacaaccc tgctggacaa
     1801 gctgatggac aggctgcgcc tgcccacgag cttgaccaca gggattgccc accggctacc
     1861 cagccttcga ccacataccc accggctcca actgcgcggc ctgcggcctt gccccatcaa
     1921 tttttttaat tttctctggg gaaaagcctc cggcctgcgg cctgcgcgct tcgcttgccg
     1981 gttggacacc aagtggaagg cgggtcaagg ctcgcgcagc gaccgcgcag cggcttggcc
     2041 ttgacgcgcc tggaacgacc caagcctatg cgagtggggg cagtcgaagg gcgaagcccg
     2101 cccgcctgcc ccccgagcct cacggcggcg agtgcggggg ttccaagggg gcagcgccac
     2161 cttgggcaag gccgaaggcc gcgcagtcga tcaacaagcc ccggaggggc cactttttgc
     2221 cggaggggga gccgcgccga aggcgtgggg gaaccccgca ggggtgccct tctttgggca
     2281 ccaaagaact agatataggg cgaaatgcga aagacttaaa aatcaacaac ttaaaaaagg
     2341 ggggtacgca acagctcatt gcggcacccc ccgcaatagc tcattgcgta ggttaaagaa
     2401 aatctgtaat tgactgccac ttttacgcaa cgcataattg ttgtcgcgct gccgaaaagt
     2461 tgcagctgat tgcgcatggt gccgcaaccg tgcggcaccc ctaccgcatg gagataagca
     2521 tggccacgca gtccagagaa atcggcattc aagccaagaa caagcccggt cactgggtgc
     2581 aaacggaacg caaagcgcat gaggcgtggg ccgggcttat tgcgaggaaa cccacggcgg
     2641 caatgctgct gcatcacctc gtggcgcaga tgggccacca gaacgccgtg gtggtcagcc
     2701 agaagacact ttccaagctc atcggacgtt ctttgcggac ggtccaatac gcagtcaagg
     2761 acttggtggc cgagcgctgg atctccgtcg tgaagctcaa cggccccggc accgtgtcgg
     2821 cctacgtggt caatgaccgc gtggcgtggg gccagccccg cgaccagttg cgcctgtcgg
     2881 tgttcagtgc cgccgtggtg gttgatcacg acgaccagga cgaatcgctg ttggggcatg
     2941 gcgacctgcg ccgcatcccg accctgtatc cgggcgagca gcaactaccg accggccccg
     3001 gcgaggagcc gcccagccag cccggcattc cgggcatgga accagacctg ccagccttga
     3061 ccgaaacgga ggaatgggaa cggcgcgggc agcagcgcct gccgatgccc gatgagccgt
     3121 gttttctgga cgatggcgag ccgttggagc cgccgacacg ggtcacgctg ccgcgccggt
     3181 agctgcagcc caaatcataa aaaatttatt tgctttgtga gcggataaca attataatag
     3241 aattcgagct cggtacccgg ggatcctcta gaaataattt tgtttaactt taagaaggag
     3301 atatacatat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     3361 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     3421 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     3481 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     3541 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     3601 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     3661 acaccctggt gaaccgcatc gag
//
