LOCUS       sequence-271492-        5209 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NanoLuciferase in bacterial cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5209)
  AUTHORS   Parag-Sharma K, O'Banion CP, Henry EC, Musicant AM, Cleveland JL, 
            Lawrence DS, Amelio AL
  TITLE     Engineered BRET-Based Biologic Light Sources Enable Spatio-Temporal 
            Control Over Diverse Optogenetic Systems.
  JOURNAL   ACS Synth Biol. 2019 Dec 13. doi: 10.1021/acssynbio.9b00277.
  PUBMED    31834783
REFERENCE   2  (bases 1 to 5209)
  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: "ACS Synth 
            Biol. 2019 Dec 13. doi: 10.1021/acssynbio.9b00277."
COMMENT     Sequence Label: pET20b-NLuc-MBP-6xHis
FEATURES             Location/Qualifiers
     source          1..5209
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             51..73
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             81..593
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             594..1691
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1548..1557
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1665..1688
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1710..1727
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1780..1798)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1794..1841
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1878..2333
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1965..1984)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2175..2196
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2359..2463
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2464..2532,2533..3324)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2682..2701)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3495..4083
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3984..4003
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4237..4254
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4269..4408
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4334..4353)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4494..4516
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4510..4701)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
       61 tttaagaagg agatatacat atggtcttca cactcgaaga tttcgttggg gactggcgac
      121 agacagccgg ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc
      181 agaatctcgg ggtgtccgta actccgatcc aaaggattgt cctgagcggt gaaaatgggc
      241 tgaagatcga catccatgtc atcatcccgt atgaaggtct gagcggcgac caaatgggcc
      301 agatcgaaaa aatttttaag gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc
      361 tgcactatgg cacactggta atcgacgggg ttacgccgaa catgatcgac tatttcggac
      421 ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt
      481 ggaacggcaa caaaattatc gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc
      541 gagtaaccat caacggagtg accggctggc ggctgtgcga acgcattctg gcgaaaatcg
      601 aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt ctcgctgaag
      661 tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat ccggataaac
      721 tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt atcttctggg
      781 cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc accccggaca
      841 aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac aacggcaagc
      901 tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa gatctgctgc
      961 cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg aaagcgaaag
     1021 gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg ctgattgctg
     1081 ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa gacgtgggcg
     1141 tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt aaaaacaaac
     1201 acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa ggcgaaacag
     1261 cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa gtgaattatg
     1321 gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga
     1381 gcgcaggtat taacgccgcc agtccgaaca aagagctggc gaaagagttc ctcgaaaact
     1441 atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag
     1501 cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc accatggaaa
     1561 acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc tggtatgccg
     1621 tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag
     1681 acgcgcagac tcgtatcacc aagctcgagc accaccacca ccaccactga gatccggctg
     1741 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     1801 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1861 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1921 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1981 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2041 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2101 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2161 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2221 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2281 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     2341 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2401 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2461 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2521 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2581 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2641 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     2701 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     2761 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     2821 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     2881 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     2941 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3001 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3061 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3121 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     3181 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3241 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3301 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3361 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3421 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3481 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3541 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3601 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3661 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3721 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3781 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3841 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3901 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3961 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4021 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4081 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4141 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4201 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4261 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     4321 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     4381 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     4441 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4501 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     4561 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     4621 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     4681 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     4741 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     4801 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     4861 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     4921 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     4981 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     5041 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     5101 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     5161 cacccgtggc caggacccaa cgctgcccga gatctcgatc ccgcgaaat
//
