LOCUS       sequence-69955-e        6411 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Shuttle vector E. coli/B.meg..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6411)
  AUTHORS   Biedendieck R, Gamer M, Jaensch L, Meyer S, Rohde M, Deckwer WD, 
            Jahn D
  TITLE     A sucrose-inducible promoter system for the intra- and extracellular
            protein production in Bacillus megaterium.
  JOURNAL   J Biotechnol. 2007 Dec 1;132(4):426-30. Epub 2007 Jul 10.
  PUBMED    17692983
REFERENCE   2  (bases 1 to 6411)
  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: "J 
            Biotechnol."; date: "2007-12-1"; volume: "132(4)"; pages: "426-30. 
            Epub 2007 Jul 10"
COMMENT     Sequence Label: pRBBm76
FEATURES             Location/Qualifiers
     source          1..6411
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(638..640,641..1354)
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="ASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(668..696)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1291..1311
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     rep_origin      complement(1536..2124)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1616..1635)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2295..3086,3087..3155))
                     /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     2918..2937
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3156..3260)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3328..3346
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(3424..4800)
                     /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(5000..6004)
                     /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"
ORIGIN
        1 ttaaggcatt atacgctctc tctttttaca aaagggagag cgtataatgc attaaaaaag
       61 ataaaaatga gtaggtaaaa agaaggaata tttttttctg aaaacatact ttagaccttt
      121 attattatca tcctaatctt tagaaaggaa aaagaataga acgttttcat gtccggctaa
      181 taaaaaaaag gtactgattt ttcaagagat tgataaaaac agaatggtcg gaaagtataa
      241 aaaacggaat agcgttttca atgctggaaa gatgaagtgt ttaatcatgt agaaagtatc
      301 aaacgcaacg tctggaaatc gtggtttgaa atgtaaaaaa gccgtacatt tttgttggaa
      361 aaacggttac ataaaaaagg ctcttacacg taaactttgg tcctagttac agctcaatga
      421 aaaagataaa aatgagaaat agctacattt ggcatgtttg actttcgtca tttatccaag
      481 taatcttttg gttgaacgga accggttctg ttttcggatt tttgatagct aattgaatta
      541 ataaatttat attctttact aacacaaaag gagaaaacat atgaaaatga aacgagttgc
      601 taagcataca actgctgcta ctagttcgaa gatctgggct agcaaaggag aagaactttt
      661 cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaatgggc acaaattttc
      721 tgtcagtgga gagggtgaag gtgatgctac atacggaaag cttaccctta aatttatttg
      781 cactactgga aaactacctg ttccatggcc aacacttgtc actactttga cctatggtgt
      841 tcaatgcttt tcccgttatc cggatcatat gaaacggcat gactttttca agagtgccat
      901 gcccgaaggt tatgtacagg aacgcactat atctttcaaa gatgacggga actacaagac
      961 gcgtgctgaa gtcaagtttg aaggtgatac ccttgttaat cgtatcgagt taaaaggtat
     1021 tgattttaaa gaagatggaa acattctcgg acacaaactc gagtacaact ataactcaca
     1081 caatgtatac atcacggcag acaaacaaaa gaatggaatc aaagctaact tcaaaattcg
     1141 ccacaacatt gaagatggat ccgttcaact agcagaccat tatcaacaaa atactccaat
     1201 tggcgatggc cctgtccttt taccagacaa ccattacctg tcgacacaat ctgccctttc
     1261 gaaagatccc aacgaaaagc gtgaccacat ggtccttctt gagtttgtaa ctgctgctgg
     1321 gattacacat ggcatggatg agctctacaa ataatgaatt cgcggccgca tgccggcggc
     1381 acctcgctaa cggattcacc actccaagaa ttggagccaa tcaattcttg cggagaactg
     1441 tgaatgcgca aaccaaccct tggcagaaca tatccatcgc gtccgccatc tccagcagcc
     1501 gcacgcggcg catctcgggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     1561 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     1621 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     1681 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     1741 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     1801 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     1861 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     1921 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     1981 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2041 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2101 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2161 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2221 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2281 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     2341 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     2401 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     2461 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     2521 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     2581 aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt
     2641 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     2701 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     2761 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     2821 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     2881 cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga
     2941 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3001 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3061 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3121 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     3181 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3241 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     3301 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa
     3361 gaattcctgt tataaaaaaa ggatcaattt tgaactctct cccaaagttg atcccttaac
     3421 gatttagaaa tccctttgag aatgtttata tacattcaag gtaaccagcc aactaatgac
     3481 aatgattcct gaaaaaagta ataacaaatt actatacaga taagttgact gatcaacttc
     3541 cataggtaac aacctttgat caagtaaggg tatggataat aaaccaccta caattgcaat
     3601 acctgttccc tctgataaaa agctggtaaa gttaagcaaa ctcattccag caccagcttc
     3661 ctgctgtttc aagctacttg aaacaattgt tgatataact gttttggtga acgaaagccc
     3721 acctaaaaca aatacgatta taattgtcat gaaccatgat gttgtttcta aaagaaagga
     3781 agcagttaaa aagctaacag aaagaaatgt aactccgatg tttaacacgt ataaaggacc
     3841 tcttctatca acaagtatcc caccaatgta gccgaaaata atgacactca ttgttccagg
     3901 gaaaataatt acacttccga tttcggcagt acttagctgg tgaacatctt tcatcatata
     3961 aggaaccata gagacaaacc ctgctactgt tccaaatata attcccccac aaagaactcc
     4021 aatcataaaa ggtatatttt tccctaatcc gggatcaaca aaaggatctg ttactttcct
     4081 gatatgtttt acaaatatca ggaatgacag cacgctaacg ataagaaaag aaatgctata
     4141 tgatgttgta aacaacataa aaaatacaat gcctacagac attagtataa ttcctttgat
     4201 atcaaaatga ccttttatcc ttacttcttt ctttaataat ttcataagaa acggaacagt
     4261 gataattgtt atcataggaa tgagtagaag ataggaccaa tgaatataat gggctatcat
     4321 tccaccaatc gctggaccga ctccttctcc catggctact atcgatccaa taagaccaaa
     4381 tgctttaccc ctattttcct ttggaatata gcgcgcaact acaaccatta cgagtgctgg
     4441 aaatgcagct gcaccagccc cttgaataaa acgagccata ataagtaagg aaaagaaaga
     4501 atggccaaca aacccaatta ccgacccgaa acaatttatt ataattccaa ataggagtaa
     4561 ccttttgatg cctaattgat cagatagctt tccatataca gctgttccaa tggaaaaggt
     4621 taacataaag gctgtgttca cccagtttgt actcgcaggt ggtttattaa aatcatttgc
     4681 aatatcaggt aatgagacgt tcaaaaccat ttcatttaat acgctaaaaa aagataaaat
     4741 gcaaagccaa attaaaattt ggttgtgtcg taaattcgat tgtgaatagg atgtattcac
     4801 atttcaccct ccaataatga gggcagacgt agtttatagg gttaatgata cgcttccctc
     4861 ttttaattga accctgttac attcattaca cttcataatt aattcctcct aaacttgatt
     4921 aaaacatttt accacatata aactaagttt taaattcagt atttcatcac ttatacaaca
     4981 atatggcccg tttgttgaac tactctttaa taaaataatt tttccgttcc caattccaca
     5041 ttgcaataat agaaaatcca tcttcatcgg ctttttcgtc atcatctgta tgaatcaaat
     5101 cgccttcttc tgtgtcatca aggtttaatt ttttatgtat ttcttttaac aaaccaccat
     5161 aggagattaa ccttttacgg tgtaaacctt cctccaaatc agacaaacgt ttcaaattct
     5221 tttcttcatc atcggtcata aaatccgtat cctttacagg atattttgca gtttcgtcaa
     5281 ttgccgattg tatatccgat ttatatttat ttttcggtcg aatcatttga acttttacat
     5341 ttggatcata gtctaatttc attgcctttt tccaaaattg aatccattgt ttttgattca
     5401 cgtagttttc tgtattctta aaataagttg gttccacaca taccaataca tgcatgtgct
     5461 gattataaga attatcttta ttatttattg tcacttccgt tgcacgcata aaaccaacaa
     5521 gatttttatt aattttttta tattgcatca ttcggcgaaa tccttgagcc atatctgaca
     5581 aactcttatt taattcttcg ccatcataaa catttttaac tgttaatgtg agaaacaacc
     5641 aacgaactgt tggcttttgt ttaataactt cagcaacaac cttttgtgac tgaatgccat
     5701 gtttcattgc tctcctccag ttgcacattg gacaaagcct ggatttacaa aaccacactc
     5761 gatacaactt tctttcgcct gtttcacgat tttgtttata ctctaatatt tcagcacaat
     5821 cttttactct ttcagccttt ttaaattcaa gaatatgcag aagttcaaag taatcaacat
     5881 tagcgatttt cttttctctc catggtctca cttttccact ttttgtcttg tccactaaaa
     5941 cccttgattt ttcatctgaa taaatgctac tattaggaca cataatatta aaagaaaccc
     6001 ccatctattt agttatttgt ttggtcactt ataactttaa cagatggggt ttttctgtgc
     6061 aaccaatttt aagggttttc aatactttaa aacacataca taccaacact tcaacgcacc
     6121 tttcagcaac taaaataaaa atgacgttat ttctatatgt atcaagataa gaaagaacaa
     6181 gttcaaaacc atcaaaaaaa gacacctttt caggtgcttt ttttatttta taaactcatt
     6241 ccctgatctc gacttcgttc tttttttacc tctcggttat gagttagttc aaattcgttc
     6301 tttttaggtt ctaaatcgtg tttttcttgg aattgtgctg ttttatcctt taccttgtct
     6361 acaaacccct taaaaacgtt tttaaaggct tttaagccgt ctgtacgttc c
//
