LOCUS       sequence-69917-e        5923 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 5923)
  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 5923)
  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+t-gfp
FEATURES             Location/Qualifiers
     source          1..5923
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1546..2550)
                     /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             2983..2994
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     RBS             3025..3033
                     /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(3071..3073,3074..3787)
                     /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(3101..3129)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3724..3744
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     rep_origin      complement(4005..4593)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4085..4104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4764..5555,5556..5624))
                     /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"
     primer_bind     5387..5406
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5625..5729)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5797..5815
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(join(5893..5923,1..1346))
                     /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
                     "
ORIGIN
        1 ttcaaggtaa ccagccaact aatgacaatg attcctgaaa aaagtaataa caaattacta
       61 tacagataag ttgactgatc aacttccata ggtaacaacc tttgatcaag taagggtatg
      121 gataataaac cacctacaat tgcaatacct gttccctctg ataaaaagct ggtaaagtta
      181 agcaaactca ttccagcacc agcttcctgc tgtttcaagc tacttgaaac aattgttgat
      241 ataactgttt tggtgaacga aagcccacct aaaacaaata cgattataat tgtcatgaac
      301 catgatgttg tttctaaaag aaaggaagca gttaaaaagc taacagaaag aaatgtaact
      361 ccgatgttta acacgtataa aggacctctt ctatcaacaa gtatcccacc aatgtagccg
      421 aaaataatga cactcattgt tccagggaaa ataattacac ttccgatttc ggcagtactt
      481 agctggtgaa catctttcat catataagga accatagaga caaaccctgc tactgttcca
      541 aatataattc ccccacaaag aactccaatc ataaaaggta tatttttccc taatccggga
      601 tcaacaaaag gatctgttac tttcctgata tgttttacaa atatcaggaa tgacagcacg
      661 ctaacgataa gaaaagaaat gctatatgat gttgtaaaca acataaaaaa tacaatgcct
      721 acagacatta gtataattcc tttgatatca aaatgacctt ttatccttac ttctttcttt
      781 aataatttca taagaaacgg aacagtgata attgttatca taggaatgag tagaagatag
      841 gaccaatgaa tataatgggc tatcattcca ccaatcgctg gaccgactcc ttctcccatg
      901 gctactatcg atccaataag accaaatgct ttacccctat tttcctttgg aatatagcgc
      961 gcaactacaa ccattacgag tgctggaaat gcagctgcac cagccccttg aataaaacga
     1021 gccataataa gtaaggaaaa gaaagaatgg ccaacaaacc caattaccga cccgaaacaa
     1081 tttattataa ttccaaatag gagtaacctt ttgatgccta attgatcaga tagctttcca
     1141 tatacagctg ttccaatgga aaaggttaac ataaaggctg tgttcaccca gtttgtactc
     1201 gcaggtggtt tattaaaatc atttgcaata tcaggtaatg agacgttcaa aaccatttca
     1261 tttaatacgc taaaaaaaga taaaatgcaa agccaaatta aaatttggtt gtgtcgtaaa
     1321 ttcgattgtg aataggatgt attcacattt caccctccaa taatgagggc agacgtagtt
     1381 tatagggtta atgatacgct tccctctttt aattgaaccc tgttacattc attacacttc
     1441 ataattaatt cctcctaaac ttgattaaaa cattttacca catataaact aagttttaaa
     1501 ttcagtattt catcacttat acaacaatat ggcccgtttg ttgaactact ctttaataaa
     1561 ataatttttc cgttcccaat tccacattgc aataatagaa aatccatctt catcggcttt
     1621 ttcgtcatca tctgtatgaa tcaaatcgcc ttcttctgtg tcatcaaggt ttaatttttt
     1681 atgtatttct tttaacaaac caccatagga gattaacctt ttacggtgta aaccttcctc
     1741 caaatcagac aaacgtttca aattcttttc ttcatcatcg gtcataaaat ccgtatcctt
     1801 tacaggatat tttgcagttt cgtcaattgc cgattgtata tccgatttat atttattttt
     1861 cggtcgaatc atttgaactt ttacatttgg atcatagtct aatttcattg cctttttcca
     1921 aaattgaatc cattgttttt gattcacgta gttttctgta ttcttaaaat aagttggttc
     1981 cacacatacc aatacatgca tgtgctgatt ataagaatta tctttattat ttattgtcac
     2041 ttccgttgca cgcataaaac caacaagatt tttattaatt tttttatatt gcatcattcg
     2101 gcgaaatcct tgagccatat ctgacaaact cttatttaat tcttcgccat cataaacatt
     2161 tttaactgtt aatgtgagaa acaaccaacg aactgttggc ttttgtttaa taacttcagc
     2221 aacaaccttt tgtgactgaa tgccatgttt cattgctctc ctccagttgc acattggaca
     2281 aagcctggat ttacaaaacc acactcgata caactttctt tcgcctgttt cacgattttg
     2341 tttatactct aatatttcag cacaatcttt tactctttca gcctttttaa attcaagaat
     2401 atgcagaagt tcaaagtaat caacattagc gattttcttt tctctccatg gtctcacttt
     2461 tccacttttt gtcttgtcca ctaaaaccct tgatttttca tctgaataaa tgctactatt
     2521 aggacacata atattaaaag aaacccccat ctatttagtt atttgtttgg tcacttataa
     2581 ctttaacaga tggggttttt ctgtgcaacc aattttaagg gttttcaata ctttaaaaca
     2641 catacatacc aacacttcaa cgcacctttc agcaactaaa ataaaaatga cgttatttct
     2701 atatgtatca agataagaaa gaacaagttc aaaaccatca aaaaaagaca ccttttcagg
     2761 tgcttttttt attttataaa ctcattccct gatctcgact tcgttctttt tttacctctc
     2821 ggttatgagt tagttcaaat tcgttctttt taggttctaa atcgtgtttt tcttggaatt
     2881 gtgctgtttt atcctttacc ttgtctacaa accccttaaa aacgttttta aaggctttta
     2941 agccgtctgt acgttcctta agatcagtat ttactggaca ctatagaagg gagaaatgtc
     3001 tccctaaatt atcaatttga tttataagga ggtgaaatgt acaatggtcc aaactagttc
     3061 gaagatctgg gctagcaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga
     3121 attagatggt gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc
     3181 tacatacgga aagcttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg
     3241 gccaacactt gtcactactt tgacctatgg tgttcaatgc ttttcccgtt atccggatca
     3301 tatgaaacgg catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac
     3361 tatatctttc aaagatgacg ggaactacaa gacgcgtgct gaagtcaagt ttgaaggtga
     3421 tacccttgtt aatcgtatcg agttaaaagg tattgatttt aaagaagatg gaaacattct
     3481 cggacacaaa ctcgagtaca actataactc acacaatgta tacatcacgg cagacaaaca
     3541 aaagaatgga atcaaagcta acttcaaaat tcgccacaac attgaagatg gatccgttca
     3601 actagcagac cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga
     3661 caaccattac ctgtcgacac aatctgccct ttcgaaagat cccaacgaaa agcgtgacca
     3721 catggtcctt cttgagtttg taactgctgc tgggattaca catggcatgg atgagcccta
     3781 caaataatga attcgcggcc gcatgctaac tagctaaccg gttacctatg ccctatctac
     3841 cttgcgtagg tagggttctt tttgttgccg gtccaagaat tggagctaat taattcttgc
     3901 ggagaactgt gaatgcgcaa accaaccctt ggcagaacat atccatcgcg tccgccatct
     3961 ccagcagccg cacgcggcgc atctcgggcc gcgttgctgg cgtttttcca taggctccgc
     4021 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4081 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4141 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4201 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4261 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4321 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4381 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4441 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4501 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4561 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4621 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4681 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4741 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4801 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4861 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4921 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4981 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5041 tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc
     5101 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5161 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5221 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5281 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5341 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca
     5401 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5461 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5521 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5581 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5641 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5701 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     5761 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     5821 cgtcttcaag aattcctgtt ataaaaaaag gatcaatttt gaactctctc ccaaagttga
     5881 tcccttaacg atttagaaat ccctttgaga atgtttatat aca
//
