LOCUS       sequence-69922-e        5887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Shuttle vector E. coli/B.meg.; gfp-expression under control of 
            RNAP-K1E-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5887)
  AUTHORS   Stammen S, Schuller F, Dietrich S, Gamer M, Biedendieck R, Jahn D
  TITLE     Application of Escherichia coli phage K1E DNA-dependent RNA 
            polymerase for in vitro RNA synthesis and in vivo protein production
            in Bacillus megaterium.
  JOURNAL   Appl Microbiol Biotechnol. 2010 Sep;88(2):529-39. doi: 
            10.1007/s00253-010-2732-y. Epub 2010 Jul 2.
  PUBMED    20596705
REFERENCE   2  (bases 1 to 5887)
  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: 
            "10.1007/s00253-010-2732-y"; journalName: "Appl Microbiol 
            Biotechnol"; date: "2010-09"; volume: "88"; issue: "2"; pages: 
            "529-39"
COMMENT     Sequence Label: pPK1E-1-gfp
FEATURES             Location/Qualifiers
     source          1..5887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     305..324
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(543..647)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     715..733
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(811..2187)
                     /codon_start=1
                     /gene="Bacillus subtilis tetL"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MNTSYSQSNLRHNQILIWLCILSFFSVLNEMVLNVSLPDIANDFN
                     KPPASTNWVNTAFMLTFSIGTAVYGKLSDQLGIKRLLLFGIIINCFGSVIGFVGHSFFS
                     LLIMARFIQGAGAAAFPALVMVVVARYIPKENRGKAFGLIGSIVAMGEGVGPAIGGMIA
                     HYIHWSYLLLIPMITIITVPFLMKLLKKEVRIKGHFDIKGIILMSVGIVFFMLFTTSYS
                     ISFLIVSVLSFLIFVKHIRKVTDPFVDPGLGKNIPFMIGVLCGGIIFGTVAGFVSMVPY
                     MMKDVHQLSTAEIGSVIIFPGTMSVIIFGYIGGILVDRRGPLYVLNIGVTFLSVSFLTA
                     SFLLETTSWFMTIIIVFVLGGLSFTKTVISTIVSSSLKQQEAGAGMSLLNFTSFLSEGT
                     GIAIVGGLLSIPLLDQRLLPMEVDQSTYLYSNLLLLFSGIIVISWLVTLNVYKHSQRDF
                     "
     CDS             complement(2387..3391)
                     /codon_start=1
                     /gene="repB"
                     /product="RepB replication protein"
                     /label=repB
                     /note="from Enterococcus faecalis plasmid pAM-alpha-1"
                     /db_xref="GI:22652809"
                     /protein_id="AAN03827.1"
                     /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
                     FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
                     QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
                     GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
                     VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
                     YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
     CDS             3824..3835
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     RBS             3866..3874
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             join(3912..3914,3915..4628)
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="ASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDEPYK"
     primer_bind     complement(3942..3970)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4565..4585
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     rep_origin      complement(4810..5398)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4890..4909)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5569..5887,1..473,474..542))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
       61 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
      121 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
      181 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
      241 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca
      301 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
      361 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
      421 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
      481 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
      541 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
      601 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
      661 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
      721 cgtatcacga ggccctttcg tcttcaagaa ttcctgttat aaaaaaagga tcaattttga
      781 actctctccc aaagttgatc ccttaacgat ttagaaatcc ctttgagaat gtttatatac
      841 attcaaggta accagccaac taatgacaat gattcctgaa aaaagtaata acaaattact
      901 atacagataa gttgactgat caacttccat aggtaacaac ctttgatcaa gtaagggtat
      961 ggataataaa ccacctacaa ttgcaatacc tgttccctct gataaaaagc tggtaaagtt
     1021 aagcaaactc attccagcac cagcttcctg ctgtttcaag ctacttgaaa caattgttga
     1081 tataactgtt ttggtgaacg aaagcccacc taaaacaaat acgattataa ttgtcatgaa
     1141 ccatgatgtt gtttctaaaa gaaaggaagc agttaaaaag ctaacagaaa gaaatgtaac
     1201 tccgatgttt aacacgtata aaggacctct tctatcaaca agtatcccac caatgtagcc
     1261 gaaaataatg acactcattg ttccagggaa aataattaca cttccgattt cggcagtact
     1321 tagctggtga acatctttca tcatataagg aaccatagag acaaaccctg ctactgttcc
     1381 aaatataatt cccccacaaa gaactccaat cataaaaggt atatttttcc ctaatccggg
     1441 atcaacaaaa ggatctgtta ctttcctgat atgttttaca aatatcagga atgacagcac
     1501 gctaacgata agaaaagaaa tgctatatga tgttgtaaac aacataaaaa atacaatgcc
     1561 tacagacatt agtataattc ctttgatatc aaaatgacct tttatcctta cttctttctt
     1621 taataatttc ataagaaacg gaacagtgat aattgttatc ataggaatga gtagaagata
     1681 ggaccaatga atataatggg ctatcattcc accaatcgct ggaccgactc cttctcccat
     1741 ggctactatc gatccaataa gaccaaatgc tttaccccta ttttcctttg gaatatagcg
     1801 cgcaactaca accattacga gtgctggaaa tgcagctgca ccagcccctt gaataaaacg
     1861 agccataata agtaaggaaa agaaagaatg gccaacaaac ccaattaccg acccgaaaca
     1921 atttattata attccaaata ggagtaacct tttgatgcct aattgatcag atagctttcc
     1981 atatacagct gttccaatgg aaaaggttaa cataaaggct gtgttcaccc agtttgtact
     2041 cgcaggtggt ttattaaaat catttgcaat atcaggtaat gagacgttca aaaccatttc
     2101 atttaatacg ctaaaaaaag ataaaatgca aagccaaatt aaaatttggt tgtgtcgtaa
     2161 attcgattgt gaataggatg tattcacatt tcaccctcca ataatgaggg cagacgtagt
     2221 ttatagggtt aatgatacgc ttccctcttt taattgaacc ctgttacatt cattacactt
     2281 cataattaat tcctcctaaa cttgattaaa acattttacc acatataaac taagttttaa
     2341 attcagtatt tcatcactta tacaacaata tggcccgttt gttgaactac tctttaataa
     2401 aataattttt ccgttcccaa ttccacattg caataataga aaatccatct tcatcggctt
     2461 tttcgtcatc atctgtatga atcaaatcgc cttcttctgt gtcatcaagg tttaattttt
     2521 tatgtatttc ttttaacaaa ccaccatagg agattaacct tttacggtgt aaaccttcct
     2581 ccaaatcaga caaacgtttc aaattctttt cttcatcatc ggtcataaaa tccgtatcct
     2641 ttacaggata ttttgcagtt tcgtcaattg ccgattgtat atccgattta tatttatttt
     2701 tcggtcgaat catttgaact tttacatttg gatcatagtc taatttcatt gcctttttcc
     2761 aaaattgaat ccattgtttt tgattcacgt agttttctgt attcttaaaa taagttggtt
     2821 ccacacatac caatacatgc atgtgctgat tataagaatt atctttatta tttattgtca
     2881 cttccgttgc acgcataaaa ccaacaagat ttttattaat ttttttatat tgcatcattc
     2941 ggcgaaatcc ttgagccata tctgacaaac tcttatttaa ttcttcgcca tcataaacat
     3001 ttttaactgt taatgtgaga aacaaccaac gaactgttgg cttttgttta ataacttcag
     3061 caacaacctt ttgtgactga atgccatgtt tcattgctct cctccagttg cacattggac
     3121 aaagcctgga tttacaaaac cacactcgat acaactttct ttcgcctgtt tcacgatttt
     3181 gtttatactc taatatttca gcacaatctt ttactctttc agccttttta aattcaagaa
     3241 tatgcagaag ttcaaagtaa tcaacattag cgattttctt ttctctccat ggtctcactt
     3301 ttccactttt tgtcttgtcc actaaaaccc ttgatttttc atctgaataa atgctactat
     3361 taggacacat aatattaaaa gaaaccccca tctatttagt tatttgtttg gtcacttata
     3421 actttaacag atggggtttt tctgtgcaac caattttaag ggttttcaat actttaaaac
     3481 acatacatac caacacttca acgcaccttt cagcaactaa aataaaaatg acgttatttc
     3541 tatatgtatc aagataagaa agaacaagtt caaaaccatc aaaaaaagac accttttcag
     3601 gtgctttttt tattttataa actcattccc tgatctcgac ttcgttcttt ttttacctct
     3661 cggttatgag ttagttcaaa ttcgttcttt ttaggttcta aatcgtgttt ttcttggaat
     3721 tgtgctgttt tatcctttac cttgtctaca aaccccttaa aaacgttttt aaaggctttt
     3781 aagccgtctg tacgttcctt aagatcagta tttactggac actatagaag ggagaaatgt
     3841 ctccctaaat tatcaatttg atttataagg aggtgaaatg tacaatggtc caaactagtt
     3901 cgaagatctg ggctagcaaa ggagaagaac ttttcactgg agttgtccca attcttgttg
     3961 aattagatgg tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg
     4021 ctacatacgg aaagcttacc cttaaattta tttgcactac tggaaaacta cctgttccat
     4081 ggccaacact tgtcactact ttgacctatg gtgttcaatg cttttcccgt tatccggatc
     4141 atatgaaacg gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaacgca
     4201 ctatatcttt caaagatgac gggaactaca agacgcgtgc tgaagtcaag tttgaaggtg
     4261 atacccttgt taatcgtatc gagttaaaag gtattgattt taaagaagat ggaaacattc
     4321 tcggacacaa actcgagtac aactataact cacacaatgt atacatcacg gcagacaaac
     4381 aaaagaatgg aatcaaagct aacttcaaaa ttcgccacaa cattgaagat ggatccgttc
     4441 aactagcaga ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag
     4501 acaaccatta cctgtcgaca caatctgccc tttcgaaaga tcccaacgaa aagcgtgacc
     4561 acatggtcct tcttgagttt gtaactgctg ctgggattac acatggcatg gatgagccct
     4621 acaaataatg aattcgcggc cgcatgccgg ctaaacctcg cgaacggatt caccggtcca
     4681 agaattggag ctaattaatt cttgcggaga actgtgaatg cgcaaaccaa cccttggcag
     4741 aacatatcca tcgcgtccgc catctccagc agccgcacgc ggcgcatctc gggccgcgtt
     4801 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4861 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4921 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4981 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5041 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5101 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5161 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5221 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     5281 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5341 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5401 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5461 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5521 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5581 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5641 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5701 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5761 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5821 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5881 tgctgca
//
