LOCUS       sequence-422517-        6890 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low 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 6890)
  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 6890)
  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: pKESaPar
FEATURES             Location/Qualifiers
     source          1..6890
                     /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      1218..1245
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1343..1362)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1354..2169
                     /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             1354..2169
                     /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(1427..1446)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2367..2802
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     CDS             complement(2839..3810)
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein of plasmid pSa"
                     /label=RepA
                     /translation="MPKNNKAPGHRINEIIKTSLALEMEDAREAGLVGYMARCLVQATM
                     PHTDPKTSNFERTNGIVTLSIMGKPSIGLPYGSMPRTLLAWICTEAVRTKDPVLNLGRS
                     QSEFLQRLGMHTDGRYTATLRNQAQRLFSSMISLAGEQGNDFGIENVVIAKRAFLFWNP
                     KRPEDRALWDSTLTLTGDFFEEVTRSPVPIRIDYLHALRQSPLAMDIYTWLTYRVFLLR
                     AKGRPFVQIPWVALQAQFGSSYGSRARNSPELDDKARERAERAALASFKYNFKKRLREV
                     LIVYPEASDCIEDDGECLRIKSTRLHVTRAPGKGARIGPPPT"
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 acgaaagact gggcctttcg
     1081 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga
     1141 tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc
     1201 caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac
     1261 tcttttgttt atttttctaa atacattcaa atatgtatcc gctcatgaac aataaaactg
     1321 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct
     1381 tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     1441 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg
     1501 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     1561 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     1621 actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta
     1681 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     1741 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     1801 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     1861 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     1921 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     1981 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     2041 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     2101 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     2161 tttttctaag aattaattca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     2221 gtcagacact agtgctactt tccgaacgac tcctgcgttg atcggaaatc cagaagcccg
     2281 agaggttgcc gcctttcggg ctttttcttt ttcaaaaaaa aaaatttata aaacgatctg
     2341 ttgcggccgc cgggttgtgg gcaaaggcgc tcgacggtgg gcaaccgctt gcggttgtcc
     2401 acgggcggag ccggtgcgcg tagcgcattg tccacaagcc aagggcgacc aataattgat
     2461 atatatattc ataattgaaa agctaattga acatactact tgctgtaact acttgccgga
     2521 gcgaggggtg tttgcaagct gttgatctga aagggctatt agcgttctca cgtgcctttt
     2581 tgattagcga tttcacgtga ccttattagc gatttcacgt actccgatta gcgatttcac
     2641 gtaccctgat tagcgatttc acgtggatag tttttggagc gggccggaaa gccccgtgaa
     2701 tcaaggcttt gcggggcatt agcggtttca cgtggataac taccctctat ccacaggctt
     2761 ccggggataa aaaagcccgc tcgacggcgg gctgttggat gggaaggctt gaccaagcca
     2821 agcgtagcgt tggcctggtc aagtcggagg ggggccgatg cgagcgccct tgccgggtgc
     2881 gcgggtgaca tgcaggcgtg tggatttgat gcgcaggcat tcgccgtcat cttcgatgca
     2941 gtcgcttgcc tcgggataga caatcaacac ttcgcgtagg cgctttttga agttgtattt
     3001 gaagctggcg agtgctgccc gctctgcccg ctctcgggcc ttatcgtcca gttcgggcga
     3061 gttgcgtgcg cggctgccat aggatgagcc gaattgcgct tgcagggcga cccaagggat
     3121 ttgcacgaag gggcggccct tggcccgcaa caggaacacg cgataggtca gccacgtgta
     3181 aatgtccatc gcaagcggag actgccgcaa ggcatgcagg tagtcgattc ggataggaac
     3241 cggtgagcgg gtgacttcct cgaagaaatc gcctgtgagg gtgagggtgc tatcccatag
     3301 cgcccgatct tctggccgct tgggattcca gaatagaaaa gcgcgcttgg caatgacgac
     3361 gttctcaatg ccgaagtcat tgccttgctc gccggcaagc gaaatcatgg atgaaaacag
     3421 gcgttgcgcc tgattgcgaa gggtggccgt gtaacggcca tcggtgtgca ttccgagcct
     3481 ttgtagaaat tccgattgcg accggccaag gttcaacacg gggtctttcg ttcgcacggc
     3541 ctcggtgcat atccaagcaa gcaaggtgcg cggcatagaa ccgtagggca ggccgatgct
     3601 cggcttgccc atgatcgaca aggtgacgat gccattggtg cgctcaaagt tgctggtctt
     3661 ggggtcggtg tggggcatgg tcgcttgcac aaggcaacgg gccatgtagc cgactaagcc
     3721 agcttcgcgg gcatcctcca tttcgagcgc gaggctcgtc ttgatgatct cgttgatacg
     3781 atggccgggg gctttgttgt tcttaggcat gttgttccct ccccggcatg gtgatggttg
     3841 gtctagtgtt tgtgggtttg atgttccggc gtttgatgaa caggcgcaag gtgtgagggc
     3901 tgacgcctaa caactcggct gcgcgacttt gcggcaagcc aaggttcacg tatgcctgta
     3961 cttcatcaat acggctgtcc agcttcaagg cgctcgattt gctgcccttg ggtcgcccga
     4021 gcgtcttgcc gcgctctctg gcgacttgta gcgcctcggt ggtacgtgcc tgaatgaaat
     4081 gccgctcgat ctgtgcagcc aagccaagca cggttgccat gatgtcgctt tgtaggctgc
     4141 cgtccatgat gatcttctgt ttggtcacat ggacgattag gccgcgctcg ctcgccgctt
     4201 tgagaatttc caaggcggcg agggcggaac cggcaatgcg cgtaatctcc ggcgtcagta
     4261 gcacgtcgcc acgctcggcc ttttcgatga ttgctccgag cttgcgcttg cgccagtcct
     4321 ttgctctgct ggcaatttct tcctcgatct gtagcggcgc gaagcctttg gcgttcgcgt
     4381 attcgagcaa accgtatttt tggttttccg ggtcttggcc gtcacgcgaa acccggagat
     4441 aggcatagta ttttggcatt tgcagggaaa acgtcagatt cggttaaaca tgcctcattc
     4501 tagcgcagat taaataggaa ttaaataccc tgttgcggta tagataaaac gttggtttgt
     4561 tctgccctat gagcgtacaa aaaaggccgg gtgagtggcc cggccttcgt ttaggtgctg
     4621 aataggattg gttctggtct gcctgcaggc gaaaaggtga gaaaagccgg gcactgcccg
     4681 gctttatttt tgctgctgcg cgttccaggc cgcccacact cgtttgacct ggctcgggct
     4741 gcatccgacc agcttggccg tcttggcaat gctcgatccg ccggagcgaa gcgtgatgat
     4801 gcggtcgtgc atgccggcgt cacgtttgcg gccggtgtag cggccggcgg ccttcgccaa
     4861 ctggacaccc tgacgttgac gctcgcgccg atcctcgtag tcgtcgcggg ccatctgcaa
     4921 ggcgagcttc aaaagcatgt cctggacgga ttccagaacg attttcgcca ctccgttcgc
     4981 ctcggcggcc agctccgaca ggtccaccac gccaggcacg gccagcttgg cccctttggc
     5041 ccggatcgac gcaaccaggc gctcggcctc ggccaacggc aagcggctga tgcggtcgat
     5101 cttctccgca acgacgactt caccaggttg caggtccgcg atcatgcgca gcagctcggg
     5161 ccggtcggcg cgtgcgccgg acgccttctc gcggtagatg ccggcgacgt agtacccggc
     5221 ggcccgcgtg gccgctacaa ggctctcctg gcgttcaaga ttctgctcgt ccgtactggc
     5281 gcgcaggtag atgcgggcga ccttcaacct tcgtccctcc ggttgttgct ctcgcgtcgc
     5341 catttccacg gctcgacggc gtgcggatcg gaccagaggc cgacgcgctt gcctcgcgcc
     5401 tcctgttcga gccgcagcat ttcagggtcg gccgcgcggc cgtggaagcg ataggcccac
     5461 gccatgccct ggtgaaccat cgcggcgttg acgttgcgcg gctgcggcgg ccggctggcc
     5521 agctccatgt tgacccacac ggtgcccagc gtgcggccgt aacggtcggt gtccttctcg
     5581 tcgaccagga cgtgccggcg gaacaccatg ccggccagcg cctggcgcgc acgttcgccg
     5641 aaggcttgcc gcttttccgg cgcgtcaatg tccaccaggc gcacgcgcac cggctgcttg
     5701 tctaccagca cgtcgatggt gtcgccgtcg atgatgcgca cgacctcgcc gcgcagctcg
     5761 gcccatgccg gcgaggcaac gaccaggacg gccagcgcgg cagcggcgcg cagcatggcg
     5821 tagcttcggc gcttcatgcg tggccccatt gctgatgatc ggggtacgcc aggtgcagca
     5881 ctgcatcgaa attggccttg cagtagccgt ccagcgccac ccgcgagccg aacgccggcg
     5941 aaaggtactc gaccaggccg ggccggtcgc ggacctcgcg ccccaggacg tggatgcgcc
     6001 ggccgcgtgt gccgtcgggt ccaggcacga aggccagcgc ctcgatgttg aagtcgatgg
     6061 atagaagttg tcggtagtgc ttggccgccc tcatcgcgtc ccccttggtc aaattgggta
     6121 tacccatttg ggcctagtct agccggcatg gcgcattaca gcaatacgca atttaaatgc
     6181 gcctagcgca ttttcccgac cttaatgcgc ctcgcgctgt agcctcacgc ccacacatgt
     6241 gctaatgtgg ttacgtgtat tttatggagg ttatccaatg agccgcctga caatcgacat
     6301 gacggaccag cagcaccaga gcctgaaagc cctggccgcc ttgcagggca agaccattaa
     6361 gcaatacgcc ctcgaacgtc tgttccccgg tgacgctgat gccgatcagg catggcagga
     6421 actgaaaacc atgctgggga accgcatcaa cgatgggctt gccggcaagg tgtccaccaa
     6481 gagcgtcggc gaaattcttg atgaagaact cagcggggat cgcgcttgac ggcctacatc
     6541 ctcacggctg aggccgaagc cgatctacgc ggcatcatcc gctacacgcg ccgggagtgg
     6601 ggcgcggcgc aggtgcgccg ctatatcgct aagctggaac agggcatagc caggcttgcc
     6661 gccggcgaag gcccgtttaa ggacatgagc gaactctttc ccgcgctgcg gatggcccgc
     6721 tgcgaacacc actacgtttt ttgcctgccg cgtgcgggcg aacccgcgtt ggtcgtggcg
     6781 atcctgcatg agcgcatgga cctcatgacg cgacttgccg acaggctcaa gggctgaccg
     6841 cgtcagacgc ccgtagcagc ccgctacggg ctttttcatg ccctgcccgc
//
