LOCUS       sequence-452404-        3224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pT7-driven expression of MGapt and deGFP for quantifying cell-free 
            RNA and protein synthesis with extended T7 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3224)
  AUTHORS   Kapasiawala M, Murray RM
  TITLE     Metabolic perturbations to an E. coli-based cell-free system reveal 
            a trade-off between transcription and translation
  JOURNAL   bioRxiv 2023.03.22.533877
REFERENCE   2  (bases 1 to 3224)
  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: "bioRxiv 
            2023.03.22.533877"
COMMENT     Sequence Label: pBEST-PT7extended-MGapt-UTR1-deGFP
FEATURES             Location/Qualifiers
     source          1..3224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     6..24
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     292..311
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        292..310
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             366..388
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(425..446)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(686..705)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1033..1054
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      1079..1126
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1267..1284)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1438..2026)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1518..1537)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2197..2988,2989..3057))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2820..2839
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3058..3162)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attctggcga atcctctgac
       61 cagccagaaa acgacctttc tgtggtgaaa ccggatgctg caattcagag cgccagcaag
      121 tgggggacag cagaagacct gaccgccgca gagtggatgt ttgacatggt gaagactatc
      181 gcaccatcag ccagaaaacc gaattttgct gggtgggcta acgatatccg cggatcccga
      241 ctgatgcgtg aacgtgacgg acgtaaccac cgcgacatgt gtgtgctgtt ctaatacgac
      301 tcactatagg gagagctagc gggatcccga ctggcgagag ccaggtaacg aatggatcca
      361 ataattttgt ttaactttaa gaaggagata taccatggag cttttcactg gcgttgttcc
      421 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
      481 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
      541 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
      601 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
      661 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
      721 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
      781 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
      841 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
      901 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
      961 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     1021 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatct aactcgagct
     1081 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttggtcg accgatgccc
     1141 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc
     1201 gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc
     1261 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     1321 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     1381 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     1441 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     1501 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     1561 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     1621 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     1681 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     1741 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     1801 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     1861 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     1921 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     1981 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2041 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     2101 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     2161 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     2221 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     2281 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     2341 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     2401 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     2461 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     2521 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     2581 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     2641 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     2701 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     2761 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga
     2821 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     2881 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     2941 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     3001 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     3061 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     3121 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     3181 gccacctgac gtctaagaaa ccattattat catgacatta acct
//
