LOCUS       sequence-331344-        5428 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 5428)
  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 5428)
  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..5428
                     /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      3106..3651
                     /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     3552..3571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3768..3785
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(join(4018..4809,4810..4878))
                     /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     4641..4660
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4879..4983)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5051..5069
                     /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 gaggaattcc
     2701 gactcaatta gttcagtcag tttcaggata ttagtcatct ctacattgat tatgagtatt
     2761 cagaaattcc ttaaatattc tgacaaatgc tctttcccta aactcccccc ataaaaaaac
     2821 ccgccgaagc gggtttttac gttatttgcg gattaacgat tactcgttat cagaaccgcc
     2881 cagacctgcg ttcagcagtt ctgccaggct ggcagatgcg tcttccggaa ttgatccgtc
     2941 gaccaaagcc cgccgaaagg cgggcttttc tgtgccggca tgataagctg tcaaacatga
     3001 gaattgcaac ttatatcgta tggggctgac ttcaggtgct acatttgaag agataaattg
     3061 cactgaaatc tagaaatatt ttatctgatt aataagatga tcttcttgag atcgttttgg
     3121 tctgcgcgta atctcttgct ctgaaaacga aaaaaccgcc ttgcagggcg gtttttcgaa
     3181 ggttctctga gctaccaact ctttgaaccg aggtaactgg cttggaggag cgcagtcacc
     3241 aaaacttgtc ctttcagttt agccttaacc ggcgcatgac ttcaagacta actcctctaa
     3301 atcaattacc agtggctgct gccagtggtg cttttgcatg tctttccggg ttggactcaa
     3361 gacgatagtt accggataag gcgcagcggt cggactgaac ggggggttcg tgcatacagt
     3421 ccagcttgga gcgaactgcc tacccggaac tgagtgtcag gcgtggaatg agacaaacgc
     3481 ggccataaca gcggaatgac accggtaaac cgaaaggcag gaacaggaga gcgcacgagg
     3541 gagccgccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccaccactga
     3601 tttgagcgtc agatttcgtg atgcttgtca ggggggcgga gcctatggaa aaacggcttt
     3661 gccgcggccc tctcacttcc ctgttaagta tcttcctggc atcttccagg aaatctccgc
     3721 cccgttcgta agccatttcc gctcgccgca gtcgaacgac cgagcgtagc gagtcagtga
     3781 gcgaggaagc ggaatatatc ctgtatcaca tattctgctg acgcaccggt gcagcctttt
     3841 ttctcctgcc acatgaagca cttcactgac accctcatca gtgccaacat agtaagccag
     3901 tatacactcc gctagggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3961 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4021 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4081 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4141 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4201 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4261 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4321 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4381 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4441 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4501 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4561 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4621 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4681 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4741 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4801 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4861 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4921 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     4981 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     5041 aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattct ggcgaatcct
     5101 ctgaccagcc agaaaacgac ctttctgtgg tgaaaccgga tgctgcaatt cagagcgcca
     5161 gcaagtgggg gacagcagaa gacctgaccg ccgcagagtg gatgtttgac atggtgaaga
     5221 ctatcgcacc atcagccaga aaaccgaatt ttgctgggtg ggctaacgat atccgcctga
     5281 tgcgtgaacg tgacggacgt aaccaccgcg acatgtgtgt gctgttccgc tgggcatgcc
     5341 aggacaactt ctggtccggt aacgtgctga gctaacaccg tgcgtgttga caattttacc
     5401 tctggcggtg ataatggttg cagctagc
//
