LOCUS       sequence-214314-        6870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  B. megaterium DSM319 pdh promoter, GFP+ with malachite green mRNA 
            aptamer in 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 6870)
  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 6870)
  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-pdh_RiboJ_GFP+_MGApt_Bba_B0015
FEATURES             Location/Qualifiers
     source          1..6870
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(1..44)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    47..66
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that start with ""ATG"""
     misc_RNA        653..702
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(781..783,784..1497)
                     /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(811..839)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1434..1454
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1578..1649
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1665..1692
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1862..3238)
                     /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(3438..4442)
                     /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(4886..4903)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5057..5645)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5137..5156)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5816..6607,6608..6676))
                     /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     6439..6458
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6677..6781)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atttcagata aaaaaaatcc ttagctttcg ctaaggatga tttctggaat tcgcggccgc
       61 ttctagaacg tctcaatctc tatgaaggcc tgtgaagttt acaggtcttt tttttggctg
      121 aaaaggaagg ttttctgaaa taaagaaaga gtttcgacat gattcacgta aaaattacaa
      181 aaggttattg ttttaaaaaa tcgtttattt gtggcaagat aggagatgtg cagacgtaaa
      241 aaaagaaaag gttattataa aacatataat acagtttgcg tctgtgtaat agtacagcat
      301 gtctttttta tgtgttttat ttcacaaact aaaaaagcgt aatttcttga tgtaaatgca
      361 ttgacgaact gttttagtgt attgtaaact aggtagcgta gtaagtgttg tattaatcat
      421 ttttgcaaaa taattaatac agtttaacga aacgttcgtt tcgcttaaac tttcctttta
      481 cgtaaatgtt tgcacagaat tcgctgggga atacatagaa atgttaagga ttagcgaatt
      541 caaaaagcta ctcgacaaaa tatgactttt acttgtccat ttgggacatt ctaaaagcgt
      601 gtatgtttgt ttgattagta aaatgaattt atacggctta aatgaagtac agctgtcacc
      661 ggatgtgctt tccggtctga tgagtccgtg aggacgaaac agcctctaca aataattttg
      721 tttaaacttg ttcacttaaa tcaaggaggt gaatgtcata tgactagttc gaagatctgg
      781 gctagcaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt
      841 gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc tacatacgga
      901 aagcttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt
      961 gtcactactt tgacctatgg tgttcaatgc ttttcccgtt atccggatca tatgaaacgg
     1021 catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc
     1081 aaagatgacg ggaactacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt
     1141 aatcgtatcg agttaaaagg tattgatttt aaagaagatg gaaacattct cggacacaaa
     1201 ctcgagtaca actataactc acacaatgta tacatcacgg cagacaaaca aaagaatgga
     1261 atcaaagcta acttcaaaat tcgccacaac attgaagatg gatccgttca actagcagac
     1321 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac
     1381 ctgtcgacac aatctgccct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt
     1441 cttgagtttg taactgctgc tgggattaca catggcatgg atgagcccta caaataatga
     1501 attcgcggcc gcatgctcga tatagcaata cggatcccga ctggcgagag ccaggtaacg
     1561 aatggatccc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     1621 ttatctgttg tttgtcggtg aacgctctct actagagtca cactggctca ccttcgggtg
     1681 ggcctttctg cgtttatatg ttaccggtcc aagtcgacca cctgacgtct aagaaaccat
     1741 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcaaga
     1801 attcctgtta taaaaaaagg atcaattttg aactctctcc caaagttgat cccttaacga
     1861 tttagaaatc cctttgagaa tgtttatata cattcaaggt aaccagccaa ctaatgacaa
     1921 tgattcctga aaaaagtaat aacaaattac tatacagata agttgactga tcaacttcca
     1981 taggtaacaa cctttgatca agtaagggta tggataataa accacctaca attgcaatac
     2041 ctgttccctc tgataaaaag ctggtaaagt taagcaaact cattccagca ccagcttcct
     2101 gctgtttcaa gctacttgaa acaattgttg atataactgt tttggtgaac gaaagcccac
     2161 ctaaaacaaa tacgattata attgtcatga accatgatgt tgtttctaaa agaaaggaag
     2221 cagttaaaaa gctaacagaa agaaatgtaa ctccgatgtt taacacgtat aaaggacctc
     2281 ttctatcaac aagtatccca ccaatgtagc cgaaaataat gacactcatt gttccaggga
     2341 aaataattac acttccgatt tcggcagtac ttagctggtg aacatctttc atcatataag
     2401 gaaccataga gacaaaccct gctactgttc caaatataat tcccccacaa agaactccaa
     2461 tcataaaagg tatatttttc cctaatccgg gatcaacaaa aggatctgtt actttcctga
     2521 tatgttttac aaatatcagg aatgacagca cgctaacgat aagaaaagaa atgctatatg
     2581 atgttgtaaa caacataaaa aatacaatgc ctacagacat tagtataatt cctttgatat
     2641 caaaatgacc ttttatcctt acttctttct ttaataattt cataagaaac ggaacagtga
     2701 taattgttat cataggaatg agtagaagat aggaccaatg aatataatgg gctatcattc
     2761 caccaatcgc tggaccgact ccttctccca tggctactat cgatccaata agaccaaatg
     2821 ctttacccct attttccttt ggaatatagc gcgcaactac aaccattacg agtgctggaa
     2881 atgcagctgc accagcccct tgaataaaac gagccataat aagtaaggaa aagaaagaat
     2941 ggccaacaaa cccaattacc gacccgaaac aatttattat aattccaaat aggagtaacc
     3001 ttttgatgcc taattgatca gatagctttc catatacagc tgttccaatg gaaaaggtta
     3061 acataaaggc tgtgttcacc cagtttgtac tcgcaggtgg tttattaaaa tcatttgcaa
     3121 tatcaggtaa tgagacgttc aaaaccattt catttaatac gctaaaaaaa gataaaatgc
     3181 aaagccaaat taaaatttgg ttgtgtcgta aattcgattg tgaataggat gtattcacat
     3241 ttcaccctcc aataatgagg gcagacgtag tttatagggt taatgatacg cttccctctt
     3301 ttaattgaac cctgttacat tcattacact tcataattaa ttcctcctaa acttgattaa
     3361 aacattttac cacatataaa ctaagtttta aattcagtat ttcatcactt atacaacaat
     3421 atggcccgtt tgttgaacta ctctttaata aaataatttt tccgttccca attccacatt
     3481 gcaataatag aaaatccatc ttcatcggct ttttcgtcat catctgtatg aatcaaatcg
     3541 ccttcttctg tgtcatcaag gtttaatttt ttatgtattt cttttaacaa accaccatag
     3601 gagattaacc ttttacggtg taaaccttcc tccaaatcag acaaacgttt caaattcttt
     3661 tcttcatcat cggtcataaa atccgtatcc tttacaggat attttgcagt ttcgtcaatt
     3721 gccgattgta tatccgattt atatttattt ttcggtcgaa tcatttgaac ttttacattt
     3781 ggatcatagt ctaatttcat tgcctttttc caaaattgaa tccattgttt ttgattcacg
     3841 tagttttctg tattcttaaa ataagttggt tccacacata ccaatacatg catgtgctga
     3901 ttataagaat tatctttatt atttattgtc acttccgttg cacgcataaa accaacaaga
     3961 tttttattaa tttttttata ttgcatcatt cggcgaaatc cttgagccat atctgacaaa
     4021 ctcttattta attcttcgcc atcataaaca tttttaactg ttaatgtgag aaacaaccaa
     4081 cgaactgttg gcttttgttt aataacttca gcaacaacct tttgtgactg aatgccatgt
     4141 ttcattgctc tcctccagtt gcacattgga caaagcctgg atttacaaaa ccacactcga
     4201 tacaactttc tttcgcctgt ttcacgattt tgtttatact ctaatatttc agcacaatct
     4261 tttactcttt cagccttttt aaattcaaga atatgcagaa gttcaaagta atcaacatta
     4321 gcgattttct tttctctcca tggtctgact tttccacttt ttgtcttgtc cactaaaacc
     4381 cttgattttt catctgaata aatgctacta ttaggacaca taatattaaa agaaaccccc
     4441 atctatttag ttatttgttt ggtcacttat aactttaaca gatggggttt ttctgtgcaa
     4501 ccaattttaa gggttttcaa tactttaaaa cacatacata ccaacacttc aacgcacctt
     4561 tcagcaacta aaataaaaat gacgttattt ctatatgtat caagataaga aagaacaagt
     4621 tcaaaaccat caaaaaaaga caccttttca ggtgcttttt ttattttata aactcattcc
     4681 ctgatctcga cttcgttctt tttttacctc tcggttatga gttagttcaa attcgttctt
     4741 tttaggttct aaatcgtgtt tttcttggaa ttgtgctgtt ttatccttta ccttgtctac
     4801 aaacccctta aaaacgtttt taaaggcttt taagccgtct gtacgttcct taaggaaggt
     4861 gtttgccaga gacgctgcag gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4921 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4981 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5041 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5101 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5161 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5221 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5281 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5341 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5401 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5461 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5521 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5581 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5641 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5701 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5761 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5821 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5881 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5941 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6001 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6061 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6121 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6181 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6241 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6301 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6361 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6421 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6481 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6541 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6601 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6661 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6721 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6781 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     6841 cctataaaaa taggcgtatc acgaggcaga
//
