LOCUS       sequence-69912-e        5956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Shuttle vector E. coli/B.meg.; gfp-expression under control of 
            RNAP-SP6-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5956)
  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 5956)
  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: pPSP6+t-gfp
FEATURES             Location/Qualifiers
     source          1..5956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(200..788)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(280..299)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(959..1750,1751..1819))
                     /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     1582..1601
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1820..1924)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1992..2010
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(2088..3464)
                     /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(3664..4668)
                     /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"
     promoter        5087..5105
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5087..5104
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             join(5222..5224,5225..5938)
                     /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(5252..5280)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5875..5895
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
ORIGIN
        1 ctaactagct aaccggttac ctatgcccta tctaccttgc gtaggtaggg ttctttttgt
       61 tgccggtcca agaattggag ctaattaatt cttgcggaga actgtgaatg cgcaaaccaa
      121 cccttggcag aacatatcca tcgcgtccgc catctccagc agccgcacgc ggcgcatctc
      181 gggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
      241 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
      301 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
      361 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
      421 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
      481 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
      541 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
      601 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
      661 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
      721 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
      781 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
      841 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
      901 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
      961 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     1021 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     1081 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     1141 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     1201 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     1261 ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     1321 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     1381 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     1441 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     1501 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     1561 cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta aaagtgctca
     1621 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     1681 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     1741 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     1801 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     1861 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     1921 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     1981 taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaattc ctgttataaa
     2041 aaaaggatca attttgaact ctctcccaaa gttgatccct taacgattta gaaatccctt
     2101 tgagaatgtt tatatacatt caaggtaacc agccaactaa tgacaatgat tcctgaaaaa
     2161 agtaataaca aattactata cagataagtt gactgatcaa cttccatagg taacaacctt
     2221 tgatcaagta agggtatgga taataaacca cctacaattg caatacctgt tccctctgat
     2281 aaaaagctgg taaagttaag caaactcatt ccagcaccag cttcctgctg tttcaagcta
     2341 cttgaaacaa ttgttgatat aactgttttg gtgaacgaaa gcccacctaa aacaaatacg
     2401 attataattg tcatgaacca tgatgttgtt tctaaaagaa aggaagcagt taaaaagcta
     2461 acagaaagaa atgtaactcc gatgtttaac acgtataaag gacctcttct atcaacaagt
     2521 atcccaccaa tgtagccgaa aataatgaca ctcattgttc cagggaaaat aattacactt
     2581 ccgatttcgg cagtacttag ctggtgaaca tctttcatca tataaggaac catagagaca
     2641 aaccctgcta ctgttccaaa tataattccc ccacaaagaa ctccaatcat aaaaggtata
     2701 tttttcccta atccgggatc aacaaaagga tctgttactt tcctgatatg ttttacaaat
     2761 atcaggaatg acagcacgct aacgataaga aaagaaatgc tatatgatgt tgtaaacaac
     2821 ataaaaaata caatgcctac agacattagt ataattcctt tgatatcaaa atgacctttt
     2881 atccttactt ctttctttaa taatttcata agaaacggaa cagtgataat tgttatcata
     2941 ggaatgagta gaagatagga ccaatgaata taatgggcta tcattccacc aatcgctgga
     3001 ccgactcctt ctcccatggc tactatcgat ccaataagac caaatgcttt acccctattt
     3061 tcctttggaa tatagcgcgc aactacaacc attacgagtg ctggaaatgc agctgcacca
     3121 gccccttgaa taaaacgagc cataataagt aaggaaaaga aagaatggcc aacaaaccca
     3181 attaccgacc cgaaacaatt tattataatt ccaaatagga gtaacctttt gatgcctaat
     3241 tgatcagata gctttccata tacagctgtt ccaatggaaa aggttaacat aaaggctgtg
     3301 ttcacccagt ttgtactcgc aggtggttta ttaaaatcat ttgcaatatc aggtaatgag
     3361 acgttcaaaa ccatttcatt taatacgcta aaaaaagata aaatgcaaag ccaaattaaa
     3421 atttggttgt gtcgtaaatt cgattgtgaa taggatgtat tcacatttca ccctccaata
     3481 atgagggcag acgtagttta tagggttaat gatacgcttc cctcttttaa ttgaaccctg
     3541 ttacattcat tacacttcat aattaattcc tcctaaactt gattaaaaca ttttaccaca
     3601 tataaactaa gttttaaatt cagtatttca tcacttatac aacaatatgg cccgtttgtt
     3661 gaactactct ttaataaaat aatttttccg ttcccaattc cacattgcaa taatagaaaa
     3721 tccatcttca tcggcttttt cgtcatcatc tgtatgaatc aaatcgcctt cttctgtgtc
     3781 atcaaggttt aattttttat gtatttcttt taacaaacca ccataggaga ttaacctttt
     3841 acggtgtaaa ccttcctcca aatcagacaa acgtttcaaa ttcttttctt catcatcggt
     3901 cataaaatcc gtatccttta caggatattt tgcagtttcg tcaattgccg attgtatatc
     3961 cgatttatat ttatttttcg gtcgaatcat ttgaactttt acatttggat catagtctaa
     4021 tttcattgcc tttttccaaa attgaatcca ttgtttttga ttcacgtagt tttctgtatt
     4081 cttaaaataa gttggttcca cacataccaa tacatgcatg tgctgattat aagaattatc
     4141 tttattattt attgtcactt ccgttgcacg cataaaacca acaagatttt tattaatttt
     4201 tttatattgc atcattcggc gaaatccttg agccatatct gacaaactct tatttaattc
     4261 ttcgccatca taaacatttt taactgttaa tgtgagaaac aaccaacgaa ctgttggctt
     4321 ttgtttaata acttcagcaa caaccttttg tgactgaatg ccatgtttca ttgctctcct
     4381 ccagttgcac attggacaaa gcctggattt acaaaaccac actcgataca actttctttc
     4441 gcctgtttca cgattttgtt tatactctaa tatttcagca caatctttta ctctttcagc
     4501 ctttttaaat tcaagaatat gcagaagttc aaagtaatca acattagcga ttttcttttc
     4561 tctccatggt ctcacttttc cactttttgt cttgtccact aaaacccttg atttttcatc
     4621 tgaataaatg ctactattag gacacataat attaaaagaa acccccatct atttagttat
     4681 ttgtttggtc acttataact ttaacagatg gggtttttct gtgcaaccaa ttttaagggt
     4741 tttcaatact ttaaaacaca tacataccaa cacttcaacg cacctttcag caactaaaat
     4801 aaaaatgacg ttatttctat atgtatcaag ataagaaaga acaagttcaa aaccatcaaa
     4861 aaaagacacc ttttcaggtg ctttttttat tttataaact cattccctga tctcgacttc
     4921 gttctttttt tacctctcgg ttatgagtta gttcaaattc gttcttttta ggttctaaat
     4981 cgtgtttttc ttggaattgt gctgttttat cctttacctt gtctacaaac cccttaaaaa
     5041 cgtttttaaa ggcttttaag ccgtctgtac gttccttaag catacgattt aggtgacact
     5101 atagaataga agtatagtgc cgttcttttg agcggcctat tactcaccag tcttcacggg
     5161 gagggctgga tagtaaaagg aggtgaaatg tacaatggtc caaactagtt cgaagatctg
     5221 ggctagcaaa ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg
     5281 tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg ctacatacgg
     5341 aaagcttacc cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact
     5401 tgtcactact ttgacctatg gtgttcaatg cttttcccgt tatccggatc atatgaaacg
     5461 gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaacgca ctatatcttt
     5521 caaagatgac gggaactaca agacgcgtgc tgaagtcaag tttgaaggtg atacccttgt
     5581 taatcgtatc gagttaaaag gtattgattt taaagaagat ggaaacattc tcggacacaa
     5641 actcgagtac aactataact cacacaatgt atacatcacg gcagacaaac aaaagaatgg
     5701 aatcaaagct aacttcaaaa ttcgccacaa cattgaagat ggatccgttc aactagcaga
     5761 ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta
     5821 cctgtcgaca caatctgccc tttcgaaaga tcccaacgaa aagcgtgacc acatggtcct
     5881 tcttgagttt gtaactgctg ctgggattac acatggcatg gatgagccct acaaataatg
     5941 aattcgcggc cgcatg
//
