LOCUS       sequence-209151-        7058 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  B. megaterium DSM319 fbp promoter, GFP+ with malachite green mRNA 
            aptamer 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 7058)
  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 7058)
  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: "Proc Natl 
            Acad Sci U S A. 2018 Apr 17. pii: 1715806115. doi: 
            10.1073/pnas.1715806115."
COMMENT     Sequence Label: pTU1-A-fbp_RiboJ_GFP+_MGApt_Bba_B0015
FEATURES             Location/Qualifiers
     source          1..7058
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        758..807
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(886..888,889..1602)
                     /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(916..944)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1539..1559
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1683..1754
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1770..1797
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1967..3343)
                     /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(3543..4547)
                     /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(4991..5008)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5162..5750)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5242..5261)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5921..6712,6713..6781))
                     /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     6544..6563
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6782..6886)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(6976..7019)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    7022..7041
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that start with ""ATG"""
ORIGIN
        1 gttcacaaat cactacattc gtacgaacac tctgcaataa ttcactttta ggttggagtg
       61 tagagataag aaaagcagcg tgatgtttta catccgctgc ttttttatat cgtaaaattc
      121 atttagtaca aggcgcccgt tgtatagcta aattgcttag gggcgggttt taaaaccgga
      181 tatactgaaa tagtgaaacg gtcagaatta gggtggataa ggtatcagca gacattaatg
      241 tgatgatttt ccctcgattt ttctcttcat ctaacaaatc caacagaaaa aatctctgaa
      301 cataaaagaa cagagattca acagaatgat gattcttata taatacaaag gatgactttt
      361 cctgtctcag aaaggtcatt tttattgtaa caccagcaaa gtcttaacgc tgtgagatat
      421 ctcacagcta gggaagaaaa aacctgctat tatatagagg tcggagtaaa ctttcttaat
      481 tatgtgcgct tctttcatat gtatcgaacc tgttgactga gatcagcagg tttttttatg
      541 ttaccagcta gctgtcatct ttaaaggggc aagatcgttt aatctttata aacaaataga
      601 ctttatacca ttttacctat acgtaaaata ggttaaaata aaggggtgag gcgtggagag
      661 aaaagagtta aataagaggt gcgttacatt atgatcacaa tttatcttac aaggttataa
      721 cggtgattat actaaatggt gaacgttgaa agtacagctg tcaccggatg tgctttccgg
      781 tctgatgagt ccgtgaggac gaaacagcct ctacaaataa ttttgtttaa acttgttcac
      841 ttaaatcaag gaggtgaatg tcatatgact agttcgaaga tctgggctag caaaggagaa
      901 gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac
      961 aaattttctg tcagtggaga gggtgaaggt gatgctacat acggaaagct tacccttaaa
     1021 tttatttgca ctactggaaa actacctgtt ccatggccaa cacttgtcac tactttgacc
     1081 tatggtgttc aatgcttttc ccgttatccg gatcatatga aacggcatga ctttttcaag
     1141 agtgccatgc ccgaaggtta tgtacaggaa cgcactatat ctttcaaaga tgacgggaac
     1201 tacaagacgc gtgctgaagt caagtttgaa ggtgataccc ttgttaatcg tatcgagtta
     1261 aaaggtattg attttaaaga agatggaaac attctcggac acaaactcga gtacaactat
     1321 aactcacaca atgtatacat cacggcagac aaacaaaaga atggaatcaa agctaacttc
     1381 aaaattcgcc acaacattga agatggatcc gttcaactag cagaccatta tcaacaaaat
     1441 actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc gacacaatct
     1501 gccctttcga aagatcccaa cgaaaagcgt gaccacatgg tccttcttga gtttgtaact
     1561 gctgctggga ttacacatgg catggatgag ccctacaaat aatgaattcg cggccgcatg
     1621 ctcgatatag caatacggat cccgactggc gagagccagg taacgaatgg atccccaggc
     1681 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     1741 cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt
     1801 atatgttacc ggtccaagtc gaccacctga cgtctaagaa accattatta tcatgacatt
     1861 aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattcc tgttataaaa
     1921 aaaggatcaa ttttgaactc tctcccaaag ttgatccctt aacgatttag aaatcccttt
     1981 gagaatgttt atatacattc aaggtaacca gccaactaat gacaatgatt cctgaaaaaa
     2041 gtaataacaa attactatac agataagttg actgatcaac ttccataggt aacaaccttt
     2101 gatcaagtaa gggtatggat aataaaccac ctacaattgc aatacctgtt ccctctgata
     2161 aaaagctggt aaagttaagc aaactcattc cagcaccagc ttcctgctgt ttcaagctac
     2221 ttgaaacaat tgttgatata actgttttgg tgaacgaaag cccacctaaa acaaatacga
     2281 ttataattgt catgaaccat gatgttgttt ctaaaagaaa ggaagcagtt aaaaagctaa
     2341 cagaaagaaa tgtaactccg atgtttaaca cgtataaagg acctcttcta tcaacaagta
     2401 tcccaccaat gtagccgaaa ataatgacac tcattgttcc agggaaaata attacacttc
     2461 cgatttcggc agtacttagc tggtgaacat ctttcatcat ataaggaacc atagagacaa
     2521 accctgctac tgttccaaat ataattcccc cacaaagaac tccaatcata aaaggtatat
     2581 ttttccctaa tccgggatca acaaaaggat ctgttacttt cctgatatgt tttacaaata
     2641 tcaggaatga cagcacgcta acgataagaa aagaaatgct atatgatgtt gtaaacaaca
     2701 taaaaaatac aatgcctaca gacattagta taattccttt gatatcaaaa tgacctttta
     2761 tccttacttc tttctttaat aatttcataa gaaacggaac agtgataatt gttatcatag
     2821 gaatgagtag aagataggac caatgaatat aatgggctat cattccacca atcgctggac
     2881 cgactccttc tcccatggct actatcgatc caataagacc aaatgcttta cccctatttt
     2941 cctttggaat atagcgcgca actacaacca ttacgagtgc tggaaatgca gctgcaccag
     3001 ccccttgaat aaaacgagcc ataataagta aggaaaagaa agaatggcca acaaacccaa
     3061 ttaccgaccc gaaacaattt attataattc caaataggag taaccttttg atgcctaatt
     3121 gatcagatag ctttccatat acagctgttc caatggaaaa ggttaacata aaggctgtgt
     3181 tcacccagtt tgtactcgca ggtggtttat taaaatcatt tgcaatatca ggtaatgaga
     3241 cgttcaaaac catttcattt aatacgctaa aaaaagataa aatgcaaagc caaattaaaa
     3301 tttggttgtg tcgtaaattc gattgtgaat aggatgtatt cacatttcac cctccaataa
     3361 tgagggcaga cgtagtttat agggttaatg atacgcttcc ctcttttaat tgaaccctgt
     3421 tacattcatt acacttcata attaattcct cctaaacttg attaaaacat tttaccacat
     3481 ataaactaag ttttaaattc agtatttcat cacttataca acaatatggc ccgtttgttg
     3541 aactactctt taataaaata atttttccgt tcccaattcc acattgcaat aatagaaaat
     3601 ccatcttcat cggctttttc gtcatcatct gtatgaatca aatcgccttc ttctgtgtca
     3661 tcaaggttta attttttatg tatttctttt aacaaaccac cataggagat taacctttta
     3721 cggtgtaaac cttcctccaa atcagacaaa cgtttcaaat tcttttcttc atcatcggtc
     3781 ataaaatccg tatcctttac aggatatttt gcagtttcgt caattgccga ttgtatatcc
     3841 gatttatatt tatttttcgg tcgaatcatt tgaactttta catttggatc atagtctaat
     3901 ttcattgcct ttttccaaaa ttgaatccat tgtttttgat tcacgtagtt ttctgtattc
     3961 ttaaaataag ttggttccac acataccaat acatgcatgt gctgattata agaattatct
     4021 ttattattta ttgtcacttc cgttgcacgc ataaaaccaa caagattttt attaattttt
     4081 ttatattgca tcattcggcg aaatccttga gccatatctg acaaactctt atttaattct
     4141 tcgccatcat aaacattttt aactgttaat gtgagaaaca accaacgaac tgttggcttt
     4201 tgtttaataa cttcagcaac aaccttttgt gactgaatgc catgtttcat tgctctcctc
     4261 cagttgcaca ttggacaaag cctggattta caaaaccaca ctcgatacaa ctttctttcg
     4321 cctgtttcac gattttgttt atactctaat atttcagcac aatcttttac tctttcagcc
     4381 tttttaaatt caagaatatg cagaagttca aagtaatcaa cattagcgat tttcttttct
     4441 ctccatggtc tgacttttcc actttttgtc ttgtccacta aaacccttga tttttcatct
     4501 gaataaatgc tactattagg acacataata ttaaaagaaa cccccatcta tttagttatt
     4561 tgtttggtca cttataactt taacagatgg ggtttttctg tgcaaccaat tttaagggtt
     4621 ttcaatactt taaaacacat acataccaac acttcaacgc acctttcagc aactaaaata
     4681 aaaatgacgt tatttctata tgtatcaaga taagaaagaa caagttcaaa accatcaaaa
     4741 aaagacacct tttcaggtgc tttttttatt ttataaactc attccctgat ctcgacttcg
     4801 ttcttttttt acctctcggt tatgagttag ttcaaattcg ttctttttag gttctaaatc
     4861 gtgtttttct tggaattgtg ctgttttatc ctttaccttg tctacaaacc ccttaaaaac
     4921 gtttttaaag gcttttaagc cgtctgtacg ttccttaagg aaggtgtttg ccagagacgc
     4981 tgcaggcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5041 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5101 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5161 ttttccacag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5221 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5281 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5341 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5401 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5461 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5521 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5581 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5641 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5701 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5761 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5821 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5881 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5941 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6001 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6061 gagagccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6121 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6181 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6241 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6301 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6361 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6421 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6481 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6541 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6601 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6661 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6721 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6781 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6841 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6901 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     6961 gtatcacgag gcagaatttc agataaaaaa aatccttagc tttcgctaag gatgatttct
     7021 ggaattcgcg gccgcttcta gaacgtctca atctctat
//
