LOCUS       sequence-69949-e        6622 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Shuttle vector E. coli/B.meg.; encoding the predicted optimal signal
            peptide for protein secretion using Bacillus megaterium under 
            control of the xylose inducible promoter .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6622)
  AUTHORS   Stammen S, Muller BK, Korneli C, Biedendieck R, Gamer M, Franco-Lara
            E, Jahn D
  TITLE     High-yield intra- and extracellular protein production using 
            Bacillus megaterium.
  JOURNAL   Appl Environ Microbiol. 2010 Jun;76(12):4037-46. doi: 
            10.1128/AEM.00431-10. Epub 2010 Apr 30.
  PUBMED    20435764
REFERENCE   2  (bases 1 to 6622)
  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: 
            "10.1128/AEM.00431-10"; journalName: "Appl Environ Microbiol"; date:
            "2010-06"; volume: "76"; issue: "12"; pages: "4037-46"
COMMENT     Sequence Label: pSPAsp-hp
FEATURES             Location/Qualifiers
     source          1..6622
                     /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             5387..5404
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     rep_origin      complement(5545..6133)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5625..5644)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6304..6622,1..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 aagatcaacg tgatataggt ttgctaacct ttgcgttcac
     3841 ttaactaact tataggggta acacttaaaa aagaatcaat aacgatagaa accgctccta
     3901 aagcaggtgc attttttcct aacgaagaag gcaatagttc acatttattg tctaaatgag
     3961 aatggactct agaagaaact tcgtttttaa tcgtatttaa aacaatggga tgagattcaa
     4021 ttatatgatt tctcaagata acagcttcta tatcaaatgt attaaggata ttggttaatc
     4081 caattccgat ataaaagcca aagttttgaa gtgcatttaa catttctaca tcatttttat
     4141 ttgcgcgttc cacaatctct tttcgagaaa tattcttttc ttctttagag agcgaagcca
     4201 gtaacgcttt ttcagaagca tataattccc aacagcctcg atttccacag ctgcatttgg
     4261 gtccattaaa atctatcgtc atatgaccca tttccccaga aaaaccctga acacctttat
     4321 acaattcgtt gttaataaca agtccagttc caattccgat attaatactg atgtaaacga
     4381 tgttttcata gttttttgtc ataccaaata ctttttcacc gtatgctcct gcattagctt
     4441 cattttcaac aaaaaccgga acattaaact cactctcaat taaaaactgc aaatctttga
     4501 tattccaatt taagttaggc atgaaaataa tttgctgatg acgatctaca aggcctggaa
     4561 cacaaattcc tattccgact agaccataag gggactcagg catatgggtt acaaaaccat
     4621 gaataagtgc aaataaaatc tcttttactt cactagcgga agaactagac aagtcagaag
     4681 tcttctcgag aataatattt ccttctaagt cggttagaat tccgttaaga tagtcgactc
     4741 ctatatcaat accaatcgag tagcctgcat tcttattaaa aacaagcatt acaggtcttc
     4801 tgccgcctct agattgccct gccccaattt caaaaataaa atctttttca agcagtgtat
     4861 ttacttgaga ggagacagta gacttgttta atcctgtaat ctcagagaga gttgccctgg
     4921 agacagggga gttcttcaaa atttcatcta atattaattt ttgattcatt ttttttacta
     4981 aagcttgatc tgcaatttga ataataacca ctcctttgtt tatccaccga actaagttgg
     5041 tgttttttga agcttgaatt agatatttaa aagtatcata tctaatatta taactaaatt
     5101 ttctaaaaaa aacattgaca taaacattta attaatatat gatgagataa agttagttta
     5161 ttggataaac aaactaactc aattaagata gttgatggat aaacttgttc acttaaatca
     5221 aggaggtgaa tgtacaatgc gtaaaagaaa aaaagcatta gctgtagcat tacttttagc
     5281 agcattaggt gcattacttc caacaacagc atctgctgat actagttcga agatctccgg
     5341 agctcccggg atccggtacc ggccgcatgc cggcctgaga ggatcgcatc accatcacca
     5401 tcactaaccg gtccaagaat tggagccaat caattcttgc ggagaactgt gaatgcgcaa
     5461 accaaccctt ggcagaacat atccatcgcg tccgccatct ccagcagccg cacgcggcgc
     5521 atctcgggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5581 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5641 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5701 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5761 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5821 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5881 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5941 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     6001 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6061 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6121 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6181 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6241 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6301 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6361 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6421 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6481 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6541 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6601 caacgttgtt gccattgctg ca
//
