LOCUS       sequence-326169-        5425 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses T7 polymerase in cell-free TXTL..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5425)
  AUTHORS   Maxwell CS, Jacobsen T, Marshall R, Noireaux V, Beisel CL
  TITLE     A detailed cell-free transcription-translation-based assay to 
            decipher CRISPR protospacer-adjacent motifs.
  JOURNAL   Methods. 2018 Jul 1;143:48-57. doi: 10.1016/j.ymeth.2018.02.016. 
            Epub 2018 Feb 24.
  PUBMED    29486239
REFERENCE   2  (bases 1 to 5425)
  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: "Methods."; 
            date: "2018-07-1"; volume: "143:48-57. doi"; pages: " 
            10.1016/j.ymeth.2018.02.016. Epub 2018 Feb 24"
COMMENT     Sequence Label: p70a-T7RNAP
FEATURES             Location/Qualifiers
     source          1..5425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             8..28
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     rep_origin      3104..3648
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     3549..3568
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3765..3782
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(join(4015..4806,4807..4875))
                     /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     4638..4657
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4876..4980)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5048..5066
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aataattttg tttaacttta agaaggagga tccaaatgaa cacgattaac atcgctaaga
       61 acgacttctc tgacatcgaa ctggctgcta tcccgttcaa cactctggct gaccattacg
      121 gtgagcgttt agctcgcgaa cagttggccc ttgagcatga gtcttacgag atgggtgaag
      181 cacgcttccg caagatgttt gagcgtcaac ttaaagctgg tgaggttgcg gataacgctg
      241 ccgccaagcc tctcatcact accctactcc ctaagatgat tgcacgcatc aacgactggt
      301 ttgaggaagt gaaagctaag cgcggcaagc gcccgacagc cttccagttc ctgcaagaaa
      361 tcaagccgga agccgtagcg tacatcacca ttaagaccac tctggcttgc ctaaccagtg
      421 ctgacaatac aaccgttcag gctgtagcaa gcgcaatcgg tcgggccatt gaggacgagg
      481 ctcgcttcgg tcgtatccgt gaccttgaag ctaagcactt caagaaaaac gttgaggaac
      541 aactcaacaa gcgcgtaggg cacgtctaca agaaagcatt tatgcaagtt gtcgaggctg
      601 acatgctctc taagggtcta ctcggtggcg aggcgtggtc ttcgtggcat aaggaagact
      661 ctattcatgt aggagtacgc tgcatcgaga tgctcattga gtcaaccgga atggttagct
      721 tacaccgcca aaatgctggc gtagtaggtc aagactctga gactatcgaa ctcgcacctg
      781 aatacgctga ggctatcgca acccgtgcag gtgcgctggc tggcatctct ccgatgttcc
      841 aaccttgcgt agttcctcct aagccgtgga ctggcattac tggtggtggc tattgggcta
      901 acggtcgtcg tcctctggcg ctggtgcgta ctcacagtaa gaaagcactg atgcgctacg
      961 aagacgttta catgcctgag gtgtacaaag cgattaacat tgcgcaaaac accgcatgga
     1021 aaatcaacaa gaaagtccta gcggtcgcca acgtaatcac caagtggaag cattgtccgg
     1081 tcgaggacat ccctgcgatt gagcgtgaag aactcccgat gaaaccggaa gacatcgaca
     1141 tgaatcctga ggctctcacc gcgtggaaac gtgctgccgc tgctgtgtac cgcaaggaca
     1201 aggctcgcaa gtctcgccgt atcagccttg agttcatgct tgagcaagcc aataagtttg
     1261 ctaaccataa ggccatctgg ttcccttaca acatggactg gcgcggtcgt gtttacgctg
     1321 tgtcaatgtt caacccgcaa ggtaacgata tgaccaaagg actgcttacg ctggcgaaag
     1381 gtaaaccaat cggtaaggaa ggttactact ggctgaaaat ccacggtgca aactgtgcgg
     1441 gtgtcgataa ggttccgttc cctgagcgca tcaagttcat tgaggaaaac cacgagaaca
     1501 tcatggcttg cgctaagtct ccactggaga acacttggtg ggctgagcaa gattctccgt
     1561 tctgcttcct tgcgttctgc tttgagtacg ctggggtaca gcaccacggc ctgagctata
     1621 actgctccct tccgctggcg tttgacgggt cttgctctgg catccagcac ttctccgcga
     1681 tgctccgaga tgaggtaggt ggtcgcgcgg ttaacttgct tcctagtgaa accgttcagg
     1741 acatctacgg gattgttgct aagaaagtca acgagattct acaagcagac gcaatcaatg
     1801 ggaccgataa cgaagtagtt accgtgaccg atgagaacac tggtgaaatc tctgagaaag
     1861 tcaagctggg cactaaggca ctggctggtc aatggctggc ttacggtgtt actcgcagtg
     1921 tgactaagcg ttcagtcatg acgctggctt acgggtccaa agagttcggc ttccgtcaac
     1981 aagtgctgga agataccatt cagccagcta ttgattccgg caagggtctg atgttcactc
     2041 agccgaatca ggctgctgga tacatggcta agctgatttg ggaatctgtg agcgtgacgg
     2101 tggtagctgc ggttgaagca atgaactggc ttaagtctgc tgctaagctg ctggctgctg
     2161 aggtcaaaga taagaagact ggagagattc ttcgcaagcg ttgcgctgtg cattgggtaa
     2221 ctcctgatgg tttccctgtg tggcaggaat acaagaagcc tattcagacg cgcttgaacc
     2281 tgatgttcct cggtcagttc cgcttacagc ctaccattaa caccaacaaa gatagcgaga
     2341 ttgatgcaca caaacaggag tctggtatcg ctcctaactt tgtacacagc caagacggta
     2401 gccaccttcg taagactgta gtgtgggcac acgagaagta cggaatcgaa tcttttgcac
     2461 tgattcacga ctccttcggt accattccgg ctgacgctgc gaacctgttc aaagcagtgc
     2521 gcgaaactat ggttgacaca tatgagtctt gtgatgtact ggctgatttc tacgaccagt
     2581 tcgctgacca gttgcacgag tctcaattgg acaaaatgcc agcacttccg gctaaaggta
     2641 acttgaacct ccgtgacatc ttagagtcgg acttcgcgtt cgcgtaactc gaggaattcg
     2701 actcaattag ttcagtcagt ttcaggatat tagtcatctc tacattgatt atgagtattc
     2761 agaaattcct taaatattct gacaaatggg gagtttaggg aaagagccca taaaaaaacc
     2821 cgccgaagcg ggtttttacg ttatttgcgg attaacgatt actcgttatc agaaccgccc
     2881 agacctgcgt tcagcagttc tgccaggctg gcagatgcgt cttccgaatt gatccgtcga
     2941 ccaaagcccg ccgaaaggcg ggcttttctg tgccggcatg ataagctgtc aaacatgaga
     3001 attacaactt atatcgtatg gggctgactt caggtgctac atttgaagag ataaattgca
     3061 ctgaaatcta gaaatatttt atctgattaa taagatgatc ttcttgagat cgttttggtc
     3121 tgcgcgtaat ctcttgctct gaaaacgaaa aaaccgcctt gcagggcggt ttttcgaagg
     3181 ttctctgagc taccaactct ttgaaccgag gtaactggct tggaggagcg cagtcaccaa
     3241 aacttgtcct ttcagtttag ccttaaccgg cgcatgactt caagactaac tcctctaaat
     3301 caattaccag tggctgctgc cagtggtgct tttgcatgtc tttccgggtt ggactcaaga
     3361 cgatagttac cggataaggc gcagcggtcg gactgaacgg ggggttcgtg catacagtcc
     3421 agcttggagc gaactgccta cccggaactg agtgtcaggc gtggaatgag acaaacgcgg
     3481 ccataacagc ggaatgacac cggtaaaccg aaaggcagga acaggagagc gcacgaggga
     3541 gccgccaggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca ccactgattt
     3601 gagcgtcaga tttcgtgatg cttgtcaggg gggcggagcc tatggaaaaa cggctttgcc
     3661 gcggccctct cacttccctg ttaagtatct tcctggcatc ttccaggaaa tctccgcccc
     3721 gttcgtaagc catttccgct cgccgcagtc gaacgaccga gcgtagcgag tcagtgagcg
     3781 aggaagcgga atatatcctg tatcacatat tctgctgacg caccggtgca gccttttttc
     3841 tcctgccaca tgaagcactt cactgacacc ctcatcagtg ccaacatagt aagccagtat
     3901 acactccgct agggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3961 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4021 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4081 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4141 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4201 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4261 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4321 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4381 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     4441 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4501 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4561 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4621 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4681 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4741 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4801 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4861 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     4921 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     4981 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     5041 ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaattctggc gaatcctctg
     5101 accagccaga aaacgacctt tctgtggtga aaccggatgc tgcaattcag agcggcagca
     5161 agtgggggac agcagaagac ctgaccgccg cagagtggat gtttgacatg gtgaagacta
     5221 tcgcaccatc agccagaaaa ccgaattttg ctgggtgggc taacgatatc cgcctgatgc
     5281 gtgaacgtga cggacgtaac caccgcgaca tgtgtgtgct gttccgctgg gcatgccagg
     5341 acaacttctg gtccggtaac gtgctgagct aacaccgtgc gtgttgacaa ttttacctct
     5401 ggcggtgata atggttgcag ctagc
//
