LOCUS       sequence-69952-e        6467 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 6467)
  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 6467)
  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: pRBBm59
FEATURES             Location/Qualifiers
     source          1..6467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(854..856,857..1570)
                     /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(884..912)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1507..1527
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     rep_origin      complement(1752..2340)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1832..1851)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2511..3302,3303..3371))
                     /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     3134..3153
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3372..3476)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3544..3562
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(3640..5016)
                     /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(5216..6220)
                     /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 acttcgttct ttttttacct ctcggttatg agttagttca aattcgttct ttttaggttc
       61 taaatcgtgt ttttcttgga attgtgctgt tttatccttt accttgtcta caaacccctt
      121 aaaaacgttt ttaaaggctt ttaagccgtc tgtacgttcc ttaagctgat tccagccgtg
      181 aaggaaaagc aactggttta gactcttaat attcaaaaaa tgcattatac gctctctctt
      241 tttacaaaag ggagagcgta taatgcatta aaaaagataa aaatgagtag gtaaaaagaa
      301 ggaatatttt tttctgaaaa catactttag acctttatta ttatcatcct aatctttaga
      361 aaggaaaaag aatagaacgt tttcatgtcc ggctaataaa aaaaaggtac tgatttttca
      421 agagattgat aaaaacagaa tggtcggaaa gtataaaaaa cggaatagcg ttttcaatgc
      481 tggaaagata aagtgtttaa tcatgtagaa agtatcaaac gcaacgtctg gaaatcgtgg
      541 tttgaaatgt aaaaaagccg tacatttttg ttggaaaaac ggttacataa aaaaggctct
      601 tacacgtaaa ctttggtcct agttacagct caatgaaaaa gataaaaatg agaaatagct
      661 acatttggca tgtttgactt tcgtcattta tccaagtaat cttttggttg aacggaaccg
      721 gttctgtttt cggatttttg atagctaatt gaattaataa atttatattc tttactaaca
      781 caaaaggaga aaacatatga aaatgaaacg agttgctaag catacaactg ctgctactag
      841 ttcgaagatc tgggctagca aaggagaaga acttttcact ggagttgtcc caattcttgt
      901 tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga
      961 tgctacatac ggaaagctta cccttaaatt tatttgcact actggaaaac tacctgttcc
     1021 atggccaaca cttgtcacta ctttgaccta tggtgttcaa tgcttttccc gttatccgga
     1081 tcatatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaacg
     1141 cactatatct ttcaaagatg acgggaacta caagacgcgt gctgaagtca agtttgaagg
     1201 tgataccctt gttaatcgta tcgagttaaa aggtattgat tttaaagaag atggaaacat
     1261 tctcggacac aaactcgagt acaactataa ctcacacaat gtatacatca cggcagacaa
     1321 acaaaagaat ggaatcaaag ctaacttcaa aattcgccac aacattgaag atggatccgt
     1381 tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc
     1441 agacaaccat tacctgtcga cacaatctgc cctttcgaaa gatcccaacg aaaagcgtga
     1501 ccacatggtc cttcttgagt ttgtaactgc tgctgggatt acacatggca tggatgagct
     1561 ctacaaataa tgaattcgcg gccgcatgcc ggcggcacct cgctaacgga ttcaccactc
     1621 caagaattgg agccaatcaa ttcttgcgga gaactgtgaa tgcgcaaacc aacccttggc
     1681 agaacatatc catcgcgtcc gccatctcca gcagccgcac gcggcgcatc tcgggccgcg
     1741 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     1801 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     1861 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     1921 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     1981 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     2041 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2101 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     2161 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     2221 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     2281 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     2341 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2401 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2461 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     2521 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2581 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     2641 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     2701 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     2761 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     2821 attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     2881 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     2941 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3001 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3061 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3121 gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     3181 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     3241 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     3301 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     3361 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     3421 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     3481 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     3541 aaaaataggc gtatcacgag gccctttcgt cttcaagaat tcctgttata aaaaaaggat
     3601 caattttgaa ctctctccca aagttgatcc cttaacgatt tagaaatccc tttgagaatg
     3661 tttatataca ttcaaggtaa ccagccaact aatgacaatg attcctgaaa aaagtaataa
     3721 caaattacta tacagataag ttgactgatc aacttccata ggtaacaacc tttgatcaag
     3781 taagggtatg gataataaac cacctacaat tgcaatacct gttccctctg ataaaaagct
     3841 ggtaaagtta agcaaactca ttccagcacc agcttcctgc tgtttcaagc tacttgaaac
     3901 aattgttgat ataactgttt tggtgaacga aagcccacct aaaacaaata cgattataat
     3961 tgtcatgaac catgatgttg tttctaaaag aaaggaagca gttaaaaagc taacagaaag
     4021 aaatgtaact ccgatgttta acacgtataa aggacctctt ctatcaacaa gtatcccacc
     4081 aatgtagccg aaaataatga cactcattgt tccagggaaa ataattacac ttccgatttc
     4141 ggcagtactt agctggtgaa catctttcat catataagga accatagaga caaaccctgc
     4201 tactgttcca aatataattc ccccacaaag aactccaatc ataaaaggta tatttttccc
     4261 taatccggga tcaacaaaag gatctgttac tttcctgata tgttttacaa atatcaggaa
     4321 tgacagcacg ctaacgataa gaaaagaaat gctatatgat gttgtaaaca acataaaaaa
     4381 tacaatgcct acagacatta gtataattcc tttgatatca aaatgacctt ttatccttac
     4441 ttctttcttt aataatttca taagaaacgg aacagtgata attgttatca taggaatgag
     4501 tagaagatag gaccaatgaa tataatgggc tatcattcca ccaatcgctg gaccgactcc
     4561 ttctcccatg gctactatcg atccaataag accaaatgct ttacccctat tttcctttgg
     4621 aatatagcgc gcaactacaa ccattacgag tgctggaaat gcagctgcac cagccccttg
     4681 aataaaacga gccataataa gtaaggaaaa gaaagaatgg ccaacaaacc caattaccga
     4741 cccgaaacaa tttattataa ttccaaatag gagtaacctt ttgatgccta attgatcaga
     4801 tagctttcca tatacagctg ttccaatgga aaaggttaac ataaaggctg tgttcaccca
     4861 gtttgtactc gcaggtggtt tattaaaatc atttgcaata tcaggtaatg agacgttcaa
     4921 aaccatttca tttaatacgc taaaaaaaga taaaatgcaa agccaaatta aaatttggtt
     4981 gtgtcgtaaa ttcgattgtg aataggatgt attcacattt caccctccaa taatgagggc
     5041 agacgtagtt tatagggtta atgatacgct tccctctttt aattgaaccc tgttacattc
     5101 attacacttc ataattaatt cctcctaaac ttgattaaaa cattttacca catataaact
     5161 aagttttaaa ttcagtattt catcacttat acaacaatat ggcccgtttg ttgaactact
     5221 ctttaataaa ataatttttc cgttcccaat tccacattgc aataatagaa aatccatctt
     5281 catcggcttt ttcgtcatca tctgtatgaa tcaaatcgcc ttcttctgtg tcatcaaggt
     5341 ttaatttttt atgtatttct tttaacaaac caccatagga gattaacctt ttacggtgta
     5401 aaccttcctc caaatcagac aaacgtttca aattcttttc ttcatcatcg gtcataaaat
     5461 ccgtatcctt tacaggatat tttgcagttt cgtcaattgc cgattgtata tccgatttat
     5521 atttattttt cggtcgaatc atttgaactt ttacatttgg atcatagtct aatttcattg
     5581 cctttttcca aaattgaatc cattgttttt gattcacgta gttttctgta ttcttaaaat
     5641 aagttggttc cacacatacc aatacatgca tgtgctgatt ataagaatta tctttattat
     5701 ttattgtcac ttccgttgca cgcataaaac caacaagatt tttattaatt tttttatatt
     5761 gcatcattcg gcgaaatcct tgagccatat ctgacaaact cttatttaat tcttcgccat
     5821 cataaacatt tttaactgtt aatgtgagaa acaaccaacg aactgttggc ttttgtttaa
     5881 taacttcagc aacaaccttt tgtgactgaa tgccatgttt cattgctctc ctccagttgc
     5941 acattggaca aagcctggat ttacaaaacc acactcgata caactttctt tcgcctgttt
     6001 cacgattttg tttatactct aatatttcag cacaatcttt tactctttca gcctttttaa
     6061 attcaagaat atgcagaagt tcaaagtaat caacattagc gattttcttt tctctccatg
     6121 gtctcacttt tccacttttt gtcttgtcca ctaaaaccct tgatttttca tctgaataaa
     6181 tgctactatt aggacacata atattaaaag aaacccccat ctatttagtt atttgtttgg
     6241 tcacttataa ctttaacaga tggggttttt ctgtgcaacc aattttaagg gttttcaata
     6301 ctttaaaaca catacatacc aacacttcaa cgcacctttc agcaactaaa ataaaaatga
     6361 cgttatttct atatgtatca agataagaaa gaacaagttc aaaaccatca aaaaaagaca
     6421 ccttttcagg tgcttttttt attttataaa ctcattccct gatctcg
//
