LOCUS       sequence-374554-        3431 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of NanoLuc via a T7 promoter in TXTL.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3431)
  AUTHORS   Sato W, Rasmussen M, Deich C, Engelhart AE, Adamala KP
  TITLE     Expanding luciferase reporter systems for cell-free protein 
            expression.
  JOURNAL   Sci Rep. 2022 Jul 7;12(1):11489. doi: 10.1038/s41598-022-15624-6.
  PUBMED    35798760
REFERENCE   2  (bases 1 to 3431)
  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: "Sci Rep."; 
            date: "2022-07-7"; volume: "12(1):11489. doi"; pages: " 
            10.1038/s41598-022-15624-6"
COMMENT     Sequence Label: pTXTL-T7max-NanoLuc
FEATURES             Location/Qualifiers
     source          1..3431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      712..1300
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1201..1220
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1379..1398
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1379..1397
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1406..1428
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1447..1959
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             1975..1998
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     rep_origin      2220..2675
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2307..2326)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2517..2538
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2742..2761)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2861..2883
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2921..2939)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3007..3111
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3112..3180,3181..3431,1..541)
                     /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     complement(3330..3349)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
       61 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
      121 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
      181 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
      241 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
      301 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
      361 gtttattgct gataaatctg gagccggtga gcgtgggtcg cgcggtatca ttgcagcact
      421 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
      481 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
      541 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
      601 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
      661 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
      721 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
      781 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
      841 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
      901 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
      961 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     1021 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     1081 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     1141 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1201 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     1261 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     1321 tttacggttc ctggcctttt gctggccttt tgctcacatg gctcgacaga tctaattcta
     1381 atacgactca ctatagggaa ataattttgt ttaactttaa gaaggagata taccggtatg
     1441 actagtatgg tcttcacact cgaagatttc gttggggact ggcgacagac agccggctac
     1501 aacctggacc aagtccttga acagggaggt gtgtccagtt tgtttcagaa tctcggggtg
     1561 tccgtaactc cgatccaaag gattgtcctg agcggtgaaa atgggctgaa gatcgacatc
     1621 catgtcatca tcccgtatga aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt
     1681 tttaaggtgg tgtaccctgt ggatgatcat cactttaagg tgatcctgca ctatggcaca
     1741 ctggtaatcg acggggttac gccgaacatg atcgactatt tcggacggcc gtatgaaggc
     1801 atcgccgtgt tcgacggcaa aaagatcact gtaacaggga ccctgtggaa cggcaacaaa
     1861 attatcgacg agcgcctgat caaccccgac ggctccctgc tgttccgagt aaccatcaac
     1921 ggagtgaccg gctggcggct gtgcgaacgc attctggcga cgcgtggagg tggccatcac
     1981 catcatcacc atcaccacgg ataactcgag caaagcccgc cgaaaggcgg gcttttctgt
     2041 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt
     2101 aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatccgggc tggcgtaata
     2161 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     2221 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2281 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2341 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2401 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2461 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2521 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2581 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2641 cgcgaatttt aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg
     2701 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
     2761 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     2821 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     2881 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     2941 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     3001 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3061 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3121 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3181 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3241 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3301 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     3361 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     3421 tcaccagtca c
//
