LOCUS       sequence-209155-        6633 bp    DNA     circular SYN 14-APR-2025
DEFINITION  B. megaterium DSM319 pdh promoter, mCherry (Bacillus codon 
            optimised) for Bacillus cell-free transcription-translation. 
            Bacillus shuttle vector backbone, colE1/AmpR (E. coli), RebB/TetA 
            (Bacillus).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6633)
  AUTHORS   Moore SJ, MacDonald JT, Wienecke S, Ishwarbhai A, Tsipa A, Aw R, 
            Kylilis N, Bell DJ, McClymont DW, Jensen K, Polizzi KM, Biedendieck 
            R, Freemont PS
  TITLE     Rapid acquisition and model-based analysis of cell-free 
            transcription-translation reactions from nonmodel bacteria.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Apr 17. pii: 1715806115. doi: 
            10.1073/pnas.1715806115.
  PUBMED    29666238
REFERENCE   2  (bases 1 to 6633)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A. 2018 Apr 17. pii: 1715806115. doi: 
            10.1073/pnas.1715806115."
COMMENT     Sequence Label: pTU1-A-pdh_RiboJ_mCherry_Bba_B0015
FEATURES             Location/Qualifiers
     source          1..6633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(866..2242)
                     /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(2442..3446)
                     /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"
     primer_bind     complement(3890..3907)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4061..4649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4141..4160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4820..5611,5612..5680))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5443..5462
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5681..5785)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(5875..5918)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    5921..5940
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that start with ""ATG"""
     misc_RNA        6527..6576
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
ORIGIN
        1 atggtatcta aaggtgaaga agataatatg gcaattatta aagaatttat gcgttttaaa
       61 gtacacatgg aaggttctgt aaatggtcat gaatttgaaa ttgaaggtga aggtgaaggt
      121 cgtccttatg aaggtacaca aacagcaaaa ttaaaagtaa caaaaggtgg tccattacca
      181 tttgcatggg atattttatc tccacaattc atgtatggtt ctaaagcgta tgtaaaacat
      241 ccagcagata ttccagatta tttaaaatta tcttttcctg aaggttttaa atgggaacgt
      301 gtaatgaatt ttgaagatgg tggtgtagta acagtaacac aagattctag tttacaagat
      361 ggtgaattta tttataaagt aaaattacgt ggtacaaatt ttccatctga tggtccagta
      421 atgcaaaaaa aaacaatggg ttgggaagca tcttctgaac gtatgtatcc tgaagatggt
      481 gcattaaaag gtgaaattaa acaacgttta aaattaaaag atggtggtca ttatgatgca
      541 gaagtaaaaa caacatataa agcaaaaaaa ccagtacaat taccaggtgc atataatgta
      601 aatattaaat tagatattac atctcataat gaagattata caattgtaga acaatatgaa
      661 cgtgcagaag gtcgtcattc tacaggtggt atggatgaat tatataaata aggatcctcg
      721 accacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
      781 cacgaggccc tttcgtcttc aagaattcct gttataaaaa aaggatcaat tttgaactct
      841 ctcccaaagt tgatccctta acgatttaga aatccctttg agaatgttta tatacattca
      901 aggtaaccag ccaactaatg acaatgattc ctgaaaaaag taataacaaa ttactataca
      961 gataagttga ctgatcaact tccataggta acaacctttg atcaagtaag ggtatggata
     1021 ataaaccacc tacaattgca atacctgttc cctctgataa aaagctggta aagttaagca
     1081 aactcattcc agcaccagct tcctgctgtt tcaagctact tgaaacaatt gttgatataa
     1141 ctgttttggt gaacgaaagc ccacctaaaa caaatacgat tataattgtc atgaaccatg
     1201 atgttgtttc taaaagaaag gaagcagtta aaaagctaac agaaagaaat gtaactccga
     1261 tgtttaacac gtataaagga cctcttctat caacaagtat cccaccaatg tagccgaaaa
     1321 taatgacact cattgttcca gggaaaataa ttacacttcc gatttcggca gtacttagct
     1381 ggtgaacatc tttcatcata taaggaacca tagagacaaa ccctgctact gttccaaata
     1441 taattccccc acaaagaact ccaatcataa aaggtatatt tttccctaat ccgggatcaa
     1501 caaaaggatc tgttactttc ctgatatgtt ttacaaatat caggaatgac agcacgctaa
     1561 cgataagaaa agaaatgcta tatgatgttg taaacaacat aaaaaataca atgcctacag
     1621 acattagtat aattcctttg atatcaaaat gaccttttat ccttacttct ttctttaata
     1681 atttcataag aaacggaaca gtgataattg ttatcatagg aatgagtaga agataggacc
     1741 aatgaatata atgggctatc attccaccaa tcgctggacc gactccttct cccatggcta
     1801 ctatcgatcc aataagacca aatgctttac ccctattttc ctttggaata tagcgcgcaa
     1861 ctacaaccat tacgagtgct ggaaatgcag ctgcaccagc cccttgaata aaacgagcca
     1921 taataagtaa ggaaaagaaa gaatggccaa caaacccaat taccgacccg aaacaattta
     1981 ttataattcc aaataggagt aaccttttga tgcctaattg atcagatagc tttccatata
     2041 cagctgttcc aatggaaaag gttaacataa aggctgtgtt cacccagttt gtactcgcag
     2101 gtggtttatt aaaatcattt gcaatatcag gtaatgagac gttcaaaacc atttcattta
     2161 atacgctaaa aaaagataaa atgcaaagcc aaattaaaat ttggttgtgt cgtaaattcg
     2221 attgtgaata ggatgtattc acatttcacc ctccaataat gagggcagac gtagtttata
     2281 gggttaatga tacgcttccc tcttttaatt gaaccctgtt acattcatta cacttcataa
     2341 ttaattcctc ctaaacttga ttaaaacatt ttaccacata taaactaagt tttaaattca
     2401 gtatttcatc acttatacaa caatatggcc cgtttgttga actactcttt aataaaataa
     2461 tttttccgtt cccaattcca cattgcaata atagaaaatc catcttcatc ggctttttcg
     2521 tcatcatctg tatgaatcaa atcgccttct tctgtgtcat caaggtttaa ttttttatgt
     2581 atttctttta acaaaccacc ataggagatt aaccttttac ggtgtaaacc ttcctccaaa
     2641 tcagacaaac gtttcaaatt cttttcttca tcatcggtca taaaatccgt atcctttaca
     2701 ggatattttg cagtttcgtc aattgccgat tgtatatccg atttatattt atttttcggt
     2761 cgaatcattt gaacttttac atttggatca tagtctaatt tcattgcctt tttccaaaat
     2821 tgaatccatt gtttttgatt cacgtagttt tctgtattct taaaataagt tggttccaca
     2881 cataccaata catgcatgtg ctgattataa gaattatctt tattatttat tgtcacttcc
     2941 gttgcacgca taaaaccaac aagattttta ttaatttttt tatattgcat cattcggcga
     3001 aatccttgag ccatatctga caaactctta tttaattctt cgccatcata aacattttta
     3061 actgttaatg tgagaaacaa ccaacgaact gttggctttt gtttaataac ttcagcaaca
     3121 accttttgtg actgaatgcc atgtttcatt gctctcctcc agttgcacat tggacaaagc
     3181 ctggatttac aaaaccacac tcgatacaac tttctttcgc ctgtttcacg attttgttta
     3241 tactctaata tttcagcaca atcttttact ctttcagcct ttttaaattc aagaatatgc
     3301 agaagttcaa agtaatcaac attagcgatt ttcttttctc tccatggtct gacttttcca
     3361 ctttttgtct tgtccactaa aacccttgat ttttcatctg aataaatgct actattagga
     3421 cacataatat taaaagaaac ccccatctat ttagttattt gtttggtcac ttataacttt
     3481 aacagatggg gtttttctgt gcaaccaatt ttaagggttt tcaatacttt aaaacacata
     3541 cataccaaca cttcaacgca cctttcagca actaaaataa aaatgacgtt atttctatat
     3601 gtatcaagat aagaaagaac aagttcaaaa ccatcaaaaa aagacacctt ttcaggtgct
     3661 ttttttattt tataaactca ttccctgatc tcgacttcgt tcttttttta cctctcggtt
     3721 atgagttagt tcaaattcgt tctttttagg ttctaaatcg tgtttttctt ggaattgtgc
     3781 tgttttatcc tttaccttgt ctacaaaccc cttaaaaacg tttttaaagg cttttaagcc
     3841 gtctgtacgt tccttaagga aggtgtttgc cagagacgct gcaggcttcc tcgctcactg
     3901 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3961 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4021 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccacagg ctccgccccc
     4081 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4141 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4201 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4261 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4321 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4381 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4441 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4501 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4561 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4621 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4681 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4741 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4801 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4861 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4921 agggcttacc atctggcccc agtgctgcaa tgataccgcg agagccacgc tcaccggctc
     4981 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5041 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5101 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5161 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5221 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5281 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5341 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5401 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5461 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5521 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5581 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5641 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5701 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5761 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     5821 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg cagaatttca
     5881 gataaaaaaa atccttagct ttcgctaagg atgatttctg gaattcgcgg ccgcttctag
     5941 aacgtctcaa tctctatgaa ggcctgtgaa gtttacaggt cttttttttg gctgaaaagg
     6001 aaggttttct gaaataaaga aagagtttcg acatgattca cgtaaaaatt acaaaaggtt
     6061 attgttttaa aaaatcgttt atttgtggca agataggaga tgtgcagacg taaaaaaaga
     6121 aaaggttatt ataaaacata taatacagtt tgcgtctgtg taatagtaca gcatgtcttt
     6181 tttatgtgtt ttatttcaca aactaaaaaa gcgtaatttc ttgatgtaaa tgcattgacg
     6241 aactgtttta gtgtattgta aactaggtag cgtagtaagt gttgtattaa tcatttttgc
     6301 aaaataatta atacagttta acgaaacgtt cgtttcgctt aaactttcct tttacgtaaa
     6361 tgtttgcaca gaattcgctg gggaatacat agaaatgtta aggattagcg aattcaaaaa
     6421 gctactcgac aaaatatgac ttttacttgt ccatttggga cattctaaaa gcgtgtatgt
     6481 ttgtttgatt agtaaaatga atttatacgg cttaaatgaa gtacagctgt caccggatgt
     6541 gctttccggt ctgatgagtc cgtgaggacg aaacagcctc tacaaataat tttgtttaaa
     6601 cttgttcact taaatcaagg aggtgaatgt cat
//
