LOCUS       sequence-209152-        6510 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  B. megaterium DSM319 gap 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 6510)
  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 6510)
  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-gap_RiboJ_GFP+_MGApt_Bba_B0015
FEATURES             Location/Qualifiers
     source          1..6510
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..717)
                     /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(31..59)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     654..674
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      798..869
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      885..912
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1082..2458)
                     /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(2658..3662)
                     /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(4106..4123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4277..4865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4357..4376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5036..5827,5828..5896))
                     /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     5659..5678
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5897..6001)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(6091..6134)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    6137..6156
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that start with ""ATG"""
     misc_RNA        6383..6432
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
ORIGIN
        1 gctagcaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt
       61 gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc tacatacgga
      121 aagcttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt
      181 gtcactactt tgacctatgg tgttcaatgc ttttcccgtt atccggatca tatgaaacgg
      241 catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc
      301 aaagatgacg ggaactacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt
      361 aatcgtatcg agttaaaagg tattgatttt aaagaagatg gaaacattct cggacacaaa
      421 ctcgagtaca actataactc acacaatgta tacatcacgg cagacaaaca aaagaatgga
      481 atcaaagcta acttcaaaat tcgccacaac attgaagatg gatccgttca actagcagac
      541 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac
      601 ctgtcgacac aatctgccct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt
      661 cttgagtttg taactgctgc tgggattaca catggcatgg atgagcccta caaataatga
      721 attcgcggcc gcatgctcga tatagcaata cggatcccga ctggcgagag ccaggtaacg
      781 aatggatccc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
      841 ttatctgttg tttgtcggtg aacgctctct actagagtca cactggctca ccttcgggtg
      901 ggcctttctg cgtttatatg ttaccggtcc aagtcgacca cctgacgtct aagaaaccat
      961 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcaaga
     1021 attcctgtta taaaaaaagg atcaattttg aactctctcc caaagttgat cccttaacga
     1081 tttagaaatc cctttgagaa tgtttatata cattcaaggt aaccagccaa ctaatgacaa
     1141 tgattcctga aaaaagtaat aacaaattac tatacagata agttgactga tcaacttcca
     1201 taggtaacaa cctttgatca agtaagggta tggataataa accacctaca attgcaatac
     1261 ctgttccctc tgataaaaag ctggtaaagt taagcaaact cattccagca ccagcttcct
     1321 gctgtttcaa gctacttgaa acaattgttg atataactgt tttggtgaac gaaagcccac
     1381 ctaaaacaaa tacgattata attgtcatga accatgatgt tgtttctaaa agaaaggaag
     1441 cagttaaaaa gctaacagaa agaaatgtaa ctccgatgtt taacacgtat aaaggacctc
     1501 ttctatcaac aagtatccca ccaatgtagc cgaaaataat gacactcatt gttccaggga
     1561 aaataattac acttccgatt tcggcagtac ttagctggtg aacatctttc atcatataag
     1621 gaaccataga gacaaaccct gctactgttc caaatataat tcccccacaa agaactccaa
     1681 tcataaaagg tatatttttc cctaatccgg gatcaacaaa aggatctgtt actttcctga
     1741 tatgttttac aaatatcagg aatgacagca cgctaacgat aagaaaagaa atgctatatg
     1801 atgttgtaaa caacataaaa aatacaatgc ctacagacat tagtataatt cctttgatat
     1861 caaaatgacc ttttatcctt acttctttct ttaataattt cataagaaac ggaacagtga
     1921 taattgttat cataggaatg agtagaagat aggaccaatg aatataatgg gctatcattc
     1981 caccaatcgc tggaccgact ccttctccca tggctactat cgatccaata agaccaaatg
     2041 ctttacccct attttccttt ggaatatagc gcgcaactac aaccattacg agtgctggaa
     2101 atgcagctgc accagcccct tgaataaaac gagccataat aagtaaggaa aagaaagaat
     2161 ggccaacaaa cccaattacc gacccgaaac aatttattat aattccaaat aggagtaacc
     2221 ttttgatgcc taattgatca gatagctttc catatacagc tgttccaatg gaaaaggtta
     2281 acataaaggc tgtgttcacc cagtttgtac tcgcaggtgg tttattaaaa tcatttgcaa
     2341 tatcaggtaa tgagacgttc aaaaccattt catttaatac gctaaaaaaa gataaaatgc
     2401 aaagccaaat taaaatttgg ttgtgtcgta aattcgattg tgaataggat gtattcacat
     2461 ttcaccctcc aataatgagg gcagacgtag tttatagggt taatgatacg cttccctctt
     2521 ttaattgaac cctgttacat tcattacact tcataattaa ttcctcctaa acttgattaa
     2581 aacattttac cacatataaa ctaagtttta aattcagtat ttcatcactt atacaacaat
     2641 atggcccgtt tgttgaacta ctctttaata aaataatttt tccgttccca attccacatt
     2701 gcaataatag aaaatccatc ttcatcggct ttttcgtcat catctgtatg aatcaaatcg
     2761 ccttcttctg tgtcatcaag gtttaatttt ttatgtattt cttttaacaa accaccatag
     2821 gagattaacc ttttacggtg taaaccttcc tccaaatcag acaaacgttt caaattcttt
     2881 tcttcatcat cggtcataaa atccgtatcc tttacaggat attttgcagt ttcgtcaatt
     2941 gccgattgta tatccgattt atatttattt ttcggtcgaa tcatttgaac ttttacattt
     3001 ggatcatagt ctaatttcat tgcctttttc caaaattgaa tccattgttt ttgattcacg
     3061 tagttttctg tattcttaaa ataagttggt tccacacata ccaatacatg catgtgctga
     3121 ttataagaat tatctttatt atttattgtc acttccgttg cacgcataaa accaacaaga
     3181 tttttattaa tttttttata ttgcatcatt cggcgaaatc cttgagccat atctgacaaa
     3241 ctcttattta attcttcgcc atcataaaca tttttaactg ttaatgtgag aaacaaccaa
     3301 cgaactgttg gcttttgttt aataacttca gcaacaacct tttgtgactg aatgccatgt
     3361 ttcattgctc tcctccagtt gcacattgga caaagcctgg atttacaaaa ccacactcga
     3421 tacaactttc tttcgcctgt ttcacgattt tgtttatact ctaatatttc agcacaatct
     3481 tttactcttt cagccttttt aaattcaaga atatgcagaa gttcaaagta atcaacatta
     3541 gcgattttct tttctctcca tggtctgact tttccacttt ttgtcttgtc cactaaaacc
     3601 cttgattttt catctgaata aatgctacta ttaggacaca taatattaaa agaaaccccc
     3661 atctatttag ttatttgttt ggtcacttat aactttaaca gatggggttt ttctgtgcaa
     3721 ccaattttaa gggttttcaa tactttaaaa cacatacata ccaacacttc aacgcacctt
     3781 tcagcaacta aaataaaaat gacgttattt ctatatgtat caagataaga aagaacaagt
     3841 tcaaaaccat caaaaaaaga caccttttca ggtgcttttt ttattttata aactcattcc
     3901 ctgatctcga cttcgttctt tttttacctc tcggttatga gttagttcaa attcgttctt
     3961 tttaggttct aaatcgtgtt tttcttggaa ttgtgctgtt ttatccttta ccttgtctac
     4021 aaacccctta aaaacgtttt taaaggcttt taagccgtct gtacgttcct taaggaaggt
     4081 gtttgccaga gacgctgcag gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4141 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4201 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4261 ccgcgttgct ggcgtttttc cacaggctcc gcccccctga cgagcatcac aaaaatcgac
     4321 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4381 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4441 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4501 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4561 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4621 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4681 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     4741 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4801 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4861 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4921 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4981 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5041 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5101 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5161 ctgcaatgat accgcgagag ccacgctcac cggctccaga tttatcagca ataaaccagc
     5221 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5281 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5341 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5401 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5461 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5521 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5581 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5641 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     5701 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5761 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5821 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5881 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5941 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6001 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     6061 cctataaaaa taggcgtatc acgaggcaga atttcagata aaaaaaatcc ttagctttcg
     6121 ctaaggatga tttctggaat tcgcggccgc ttctagaacg tctcaatctc tatagagtga
     6181 atgattgcat tcactctttt tttattggga atatttaagg gcttctaaaa aagaatgtgt
     6241 taaatatctg ttctcgctat atacatatag taaaacttaa ttaaaataat ttattaaaaa
     6301 aaattaatat tattcacttt ttaaatattt atgttatact attcaatata aaaagttaaa
     6361 tagtataaca catatagtac agctgtcacc ggatgtgctt tccggtctga tgagtccgtg
     6421 aggacgaaac agcctctaca aataattttg tttaaacttg ttcacttaaa tcaaggaggt
     6481 gaatgtcata tgactagttc gaagatctgg
//
