LOCUS       sequence-422515-        4648 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..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4648)
  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 4648)
  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: pKESa
FEATURES             Location/Qualifiers
     source          1..4648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     248..269
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(353..370)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      688..715
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(813..832)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             824..1639
                     /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             824..1639
                     /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(897..916)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1837..2272
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     CDS             complement(2309..3280)
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein of plasmid pSa"
                     /label=RepA
                     /translation="MPKNNKAPGHRINEIIKTSLALEMEDAREAGLVGYMARCLVQATM
                     PHTDPKTSNFERTNGIVTLSIMGKPSIGLPYGSMPRTLLAWICTEAVRTKDPVLNLGRS
                     QSEFLQRLGMHTDGRYTATLRNQAQRLFSSMISLAGEQGNDFGIENVVIAKRAFLFWNP
                     KRPEDRALWDSTLTLTGDFFEEVTRSPVPIRIDYLHALRQSPLAMDIYTWLTYRVFLLR
                     AKGRPFVQIPWVALQAQFGSSYGSRARNSPELDDKARERAERAALASFKYNFKKRLREV
                     LIVYPEASDCIEDDGECLRIKSTRLHVTRAPGKGARIGPPPT"
     promoter        join(4129..4143,4144..4149,4150..4166,4167..4173)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    4149..4165
                     /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             4213..4235
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(4243..4245,4246..4248,4249..4648,1..314)
                     /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(4243..4245,4246..4248,4249..4648,1..311)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4288..4309)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4549..4568)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
       61 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
      121 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
      181 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
      241 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
      301 agctgtacaa gtaaaagctt ggctgttttg gcggatgaga gaagattttc agcctgatac
      361 agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg
      421 cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta
      481 gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaagact
      541 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
      601 cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc
      661 cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt
      721 ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc gctcatgaac
      781 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg
      841 ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
      901 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa
      961 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
     1021 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca
     1081 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc
     1141 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
     1201 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt
     1261 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt
     1321 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt
     1381 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
     1441 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     1501 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     1561 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     1621 gctcgatgag tttttctaag aattaattca tgaccaaaat cccttaacgt gagttttcgt
     1681 tccactgagc gtcagacact agtgctactt tccgaacgac tcctgcgttg atcggaaatc
     1741 cagaagcccg agaggttgcc gcctttcggg ctttttcttt ttcaaaaaaa aaaatttata
     1801 aaacgatctg ttgcggccgc cgggttgtgg gcaaaggcgc tcgacggtgg gcaaccgctt
     1861 gcggttgtcc acgggcggag ccggtgcgcg tagcgcattg tccacaagcc aagggcgacc
     1921 aataattgat atatatattc ataattgaaa agctaattga acatactact tgctgtaact
     1981 acttgccgga gcgaggggtg tttgcaagct gttgatctga aagggctatt agcgttctca
     2041 cgtgcctttt tgattagcga tttcacgtga ccttattagc gatttcacgt actccgatta
     2101 gcgatttcac gtaccctgat tagcgatttc acgtggatag tttttggagc gggccggaaa
     2161 gccccgtgaa tcaaggcttt gcggggcatt agcggtttca cgtggataac taccctctat
     2221 ccacaggctt ccggggataa aaaagcccgc tcgacggcgg gctgttggat gggaaggctt
     2281 gaccaagcca agcgtagcgt tggcctggtc aagtcggagg ggggccgatg cgagcgccct
     2341 tgccgggtgc gcgggtgaca tgcaggcgtg tggatttgat gcgcaggcat tcgccgtcat
     2401 cttcgatgca gtcgcttgcc tcgggataga caatcaacac ttcgcgtagg cgctttttga
     2461 agttgtattt gaagctggcg agtgctgccc gctctgcccg ctctcgggcc ttatcgtcca
     2521 gttcgggcga gttgcgtgcg cggctgccat aggatgagcc gaattgcgct tgcagggcga
     2581 cccaagggat ttgcacgaag gggcggccct tggcccgcaa caggaacacg cgataggtca
     2641 gccacgtgta aatgtccatc gcaagcggag actgccgcaa ggcatgcagg tagtcgattc
     2701 ggataggaac cggtgagcgg gtgacttcct cgaagaaatc gcctgtgagg gtgagggtgc
     2761 tatcccatag cgcccgatct tctggccgct tgggattcca gaatagaaaa gcgcgcttgg
     2821 caatgacgac gttctcaatg ccgaagtcat tgccttgctc gccggcaagc gaaatcatgg
     2881 atgaaaacag gcgttgcgcc tgattgcgaa gggtggccgt gtaacggcca tcggtgtgca
     2941 ttccgagcct ttgtagaaat tccgattgcg accggccaag gttcaacacg gggtctttcg
     3001 ttcgcacggc ctcggtgcat atccaagcaa gcaaggtgcg cggcatagaa ccgtagggca
     3061 ggccgatgct cggcttgccc atgatcgaca aggtgacgat gccattggtg cgctcaaagt
     3121 tgctggtctt ggggtcggtg tggggcatgg tcgcttgcac aaggcaacgg gccatgtagc
     3181 cgactaagcc agcttcgcgg gcatcctcca tttcgagcgc gaggctcgtc ttgatgatct
     3241 cgttgatacg atggccgggg gctttgttgt tcttaggcat gttgttccct ccccggcatg
     3301 gtgatggttg gtctagtgtt tgtgggtttg atgttccggc gtttgatgaa caggcgcaag
     3361 gtgtgagggc tgacgcctaa caactcggct gcgcgacttt gcggcaagcc aaggttcacg
     3421 tatgcctgta cttcatcaat acggctgtcc agcttcaagg cgctcgattt gctgcccttg
     3481 ggtcgcccga gcgtcttgcc gcgctctctg gcgacttgta gcgcctcggt ggtacgtgcc
     3541 tgaatgaaat gccgctcgat ctgtgcagcc aagccaagca cggttgccat gatgtcgctt
     3601 tgtaggctgc cgtccatgat gatcttctgt ttggtcacat ggacgattag gccgcgctcg
     3661 ctcgccgctt tgagaatttc caaggcggcg agggcggaac cggcaatgcg cgtaatctcc
     3721 ggcgtcagta gcacgtcgcc acgctcggcc ttttcgatga ttgctccgag cttgcgcttg
     3781 cgccagtcct ttgctctgct ggcaatttct tcctcgatct gtagcggcgc gaagcctttg
     3841 gcgttcgcgt attcgagcaa accgtatttt tggttttccg ggtcttggcc gtcacgcgaa
     3901 acccggagat aggcatagta ttttggcatt tgcagggaaa acgtcagatt cggttaaaca
     3961 tgcctcattc tagcgcagat taaataggaa ttaaataccc tgttgcggta tagataaaac
     4021 gttggtttgt tctgccctat gagcgtacaa aaaaggccgg gtgagtggcc cggccttcgt
     4081 ttaggtgctg aataggattg gttctggtct gcctgcaggg ccgccaaatc ataaaaaatt
     4141 tatttgcttt gtgagcggat aacaattata atagaattcg agctcggtac ccggggatcc
     4201 tctagaaata attttgttta actttaagaa ggagatatac atatggtgag caagggcgag
     4261 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     4321 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     4381 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     4441 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     4501 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     4561 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     4621 aagggcatcg acttcaagga ggacggca
//
