LOCUS       sequence-422937-        5929 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 and a
            RP4 partitioning system to increase segregational stability..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5929)
  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 5929)
  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: pKERepPar
FEATURES             Location/Qualifiers
     source          1..5929
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(11..25,26..31,32..48,49..55)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    31..47
                     /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             95..117
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(125..127,128..130,131..844)
                     /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(125..127,128..130,131..841)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(170..191)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(431..450)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     778..799
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(883..900)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1053..1139
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1231..1258
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1356..1375)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1367..2182
                     /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             1367..2182
                     /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(1440..1459)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2247..3018
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             3019..3681
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
ORIGIN
        1 ggccgccaaa tcataaaaaa tttatttgct ttgtgagcgg ataacaatta taatagaatt
       61 cgagctcggt acccggggat cctctagaaa taattttgtt taactttaag aaggagatat
      121 acatatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      181 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      241 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      301 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
      361 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
      421 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
      481 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
      541 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
      601 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
      661 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
      721 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
      781 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
      841 gtaaaagctt ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc
      901 agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc
      961 acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc
     1021 tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag
     1081 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
     1141 cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc
     1201 cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg
     1261 cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1321 aataataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     1381 acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg
     1441 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
     1501 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     1561 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     1621 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac
     1681 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     1741 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     1801 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     1861 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     1921 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     1981 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     2041 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     2101 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     2161 gatgctcgat gagtttttct aagaattaat tcatgaccaa aatcccttaa cgtgagtttt
     2221 cgttccactg agcgtcagac actagtgcgg ccaccggctg gctcgcttcg ctcggcccgt
     2281 ggacaaccct gctggacaag ctgatggaca ggctgcgcct gcccacgagc ttgaccacag
     2341 ggattgccca ccggctaccc agccttcgac cacataccca ccggctccaa ctgcgcggcc
     2401 tgcggccttg ccccatcaat ttttttaatt ttctctgggg aaaagcctcc ggcctgcggc
     2461 ctgcgcgctt cgcttgccgg ttggacacca agtggaaggc gggtcaaggc tcgcgcagcg
     2521 accgcgcagc ggcttggcct tgacgcgcct ggaacgaccc aagcctatgc gagtgggggc
     2581 agtcgaaggg cgaagcccgc ccgcctgccc cccgagcctc acggcggcga gtgcgggggt
     2641 tccaaggggg cagcgccacc ttgggcaagg ccgaaggccg cgcagtcgat caacaagccc
     2701 cggaggggcc actttttgcc ggagggggag ccgcgccgaa ggcgtggggg aaccccgcag
     2761 gggtgccctt ctttgggcac caaagaacta gatatagggc gaaatgcgaa agacttaaaa
     2821 atcaacaact taaaaaaggg gggtacgcaa cagctcattg cggcaccccc cgcaatagct
     2881 cattgcgtag gttaaagaaa atctgtaatt gactgccact tttacgcaac gcataattgt
     2941 tgtcgcgctg ccgaaaagtt gcagctgatt gcgcatggtg ccgcaaccgt gcggcacccc
     3001 taccgcatgg agataagcat ggccacgcag tccagagaaa tcggcattca agccaagaac
     3061 aagcccggtc actgggtgca aacggaacgc aaagcgcatg aggcgtgggc cgggcttatt
     3121 gcgaggaaac ccacggcggc aatgctgctg catcacctcg tggcgcagat gggccaccag
     3181 aacgccgtgg tggtcagcca gaagacactt tccaagctca tcggacgttc tttgcggacg
     3241 gtccaatacg cagtcaagga cttggtggcc gagcgctgga tctccgtcgt gaagctcaac
     3301 ggccccggca ccgtgtcggc ctacgtggtc aatgaccgcg tggcgtgggg ccagccccgc
     3361 gaccagttgc gcctgtcggt gttcagtgcc gccgtggtgg ttgatcacga cgaccaggac
     3421 gaatcgctgt tggggcatgg cgacctgcgc cgcatcccga ccctgtatcc gggcgagcag
     3481 caactaccga ccggccccgg cgaggagccg cccagccagc ccggcattcc gggcatggaa
     3541 ccagacctgc cagccttgac cgaaacggag gaatgggaac ggcgcgggca gcagcgcctg
     3601 ccgatgcccg atgagccgtg ttttctggac gatggcgagc cgttggagcc gccgacacgg
     3661 gtcacgctgc cgcgccggta gctgcaggcg aaaaggtgag aaaagccggg cactgcccgg
     3721 ctttattttt gctgctgcgc gttccaggcc gcccacactc gtttgacctg gctcgggctg
     3781 catccgacca gcttggccgt cttggcaatg ctcgatccgc cggagcgaag cgtgatgatg
     3841 cggtcgtgca tgccggcgtc acgtttgcgg ccggtgtagc ggccggcggc cttcgccaac
     3901 tggacaccct gacgttgacg ctcgcgccga tcctcgtagt cgtcgcgggc catctgcaag
     3961 gcgagcttca aaagcatgtc ctggacggat tccagaacga ttttcgccac tccgttcgcc
     4021 tcggcggcca gctccgacag gtccaccacg ccaggcacgg ccagcttggc ccctttggcc
     4081 cggatcgacg caaccaggcg ctcggcctcg gccaacggca agcggctgat gcggtcgatc
     4141 ttctccgcaa cgacgacttc accaggttgc aggtccgcga tcatgcgcag cagctcgggc
     4201 cggtcggcgc gtgcgccgga cgccttctcg cggtagatgc cggcgacgta gtacccggcg
     4261 gcccgcgtgg ccgctacaag gctctcctgg cgttcaagat tctgctcgtc cgtactggcg
     4321 cgcaggtaga tgcgggcgac cttcaacctt cgtccctccg gttgttgctc tcgcgtcgcc
     4381 atttccacgg ctcgacggcg tgcggatcgg accagaggcc gacgcgcttg cctcgcgcct
     4441 cctgttcgag ccgcagcatt tcagggtcgg ccgcgcggcc gtggaagcga taggcccacg
     4501 ccatgccctg gtgaaccatc gcggcgttga cgttgcgcgg ctgcggcggc cggctggcca
     4561 gctccatgtt gacccacacg gtgcccagcg tgcggccgta acggtcggtg tccttctcgt
     4621 cgaccaggac gtgccggcgg aacaccatgc cggccagcgc ctggcgcgca cgttcgccga
     4681 aggcttgccg cttttccggc gcgtcaatgt ccaccaggcg cacgcgcacc ggctgcttgt
     4741 ctaccagcac gtcgatggtg tcgccgtcga tgatgcgcac gacctcgccg cgcagctcgg
     4801 cccatgccgg cgaggcaacg accaggacgg ccagcgcggc agcggcgcgc agcatggcgt
     4861 agcttcggcg cttcatgcgt ggccccattg ctgatgatcg gggtacgcca ggtgcagcac
     4921 tgcatcgaaa ttggccttgc agtagccgtc cagcgccacc cgcgagccga acgccggcga
     4981 aaggtactcg accaggccgg gccggtcgcg gacctcgcgc cccaggacgt ggatgcgccg
     5041 gccgcgtgtg ccgtcgggtc caggcacgaa ggccagcgcc tcgatgttga agtcgatgga
     5101 tagaagttgt cggtagtgct tggccgccct catcgcgtcc cccttggtca aattgggtat
     5161 acccatttgg gcctagtcta gccggcatgg cgcattacag caatacgcaa tttaaatgcg
     5221 cctagcgcat tttcccgacc ttaatgcgcc tcgcgctgta gcctcacgcc cacacatgtg
     5281 ctaatgtggt tacgtgtatt ttatggaggt tatccaatga gccgcctgac aatcgacatg
     5341 acggaccagc agcaccagag cctgaaagcc ctggccgcct tgcagggcaa gaccattaag
     5401 caatacgccc tcgaacgtct gttccccggt gacgctgatg ccgatcaggc atggcaggaa
     5461 ctgaaaacca tgctggggaa ccgcatcaac gatgggcttg ccggcaaggt gtccaccaag
     5521 agcgtcggcg aaattcttga tgaagaactc agcggggatc gcgcttgacg gcctacatcc
     5581 tcacggctga ggccgaagcc gatctacgcg gcatcatccg ctacacgcgc cgggagtggg
     5641 gcgcggcgca ggtgcgccgc tatatcgcta agctggaaca gggcatagcc aggcttgccg
     5701 ccggcgaagg cccgtttaag gacatgagcg aactctttcc cgcgctgcgg atggcccgct
     5761 gcgaacacca ctacgttttt tgcctgccgc gtgcgggcga acccgcgttg gtcgtggcga
     5821 tcctgcatga gcgcatggac ctcatgacgc gacttgccga caggctcaag ggctgaccgc
     5881 gtcagacgcc cgtagcagcc cgctacgggc tttttcatgc cctgcccgc
//
