LOCUS       sequence-271490-        4831 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the GpNLuc LumiFluor in bacterial cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4831)
  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 4831)
  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-GpNLuc-6xHis
FEATURES             Location/Qualifiers
     source          1..4831
                     /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             join(81..83,84..86,87..797)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(81..83,84..86,87..797)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(126..147)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(387..406)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     734..755
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             813..1325
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             1332..1349
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1402..1420)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1416..1463
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1500..1955
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1587..1606)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1797..1818
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1981..2085
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2086..2154,2155..2946)
                     /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(2304..2323)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3117..3705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3606..3625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3859..3876
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3891..4030
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3956..3975)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4116..4138
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4132..4323)
                     /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 atggtgagca agggcgagga gctgttcacc ggggtggtgc
      121 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
      181 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
      241 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
      301 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
      361 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
      421 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
      481 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
      541 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
      601 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
      661 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
      721 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
      781 tggacgagct gtacaaggat atctccggag gtatggtctt cacactcgaa gatttcgttg
      841 gggactggcg acagacagcc ggctacaacc tggaccaagt ccttgaacag ggaggtgtgt
      901 ccagtttgtt tcagaatctc ggggtgtccg taactccgat ccaaaggatt gtcctgagcg
      961 gtgaaaatgg gctgaagatc gacatccatg tcatcatccc gtatgaaggt ctgagcggcg
     1021 accaaatggg ccagatcgaa aaaattttta aggtggtgta ccctgtggat gatcatcact
     1081 ttaaggtgat cctgcactat ggcacactgg taatcgacgg ggttacgccg aacatgatcg
     1141 actatttcgg acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa
     1201 cagggaccct gtggaacggc aacaaaatta tcgacgagcg cctgatcaac cccgacggct
     1261 ccctgctgtt ccgagtaacc atcaacggag tgaccggctg gcggctgtgc gaacgcattc
     1321 tggcgctcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
     1381 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     1441 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     1501 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1561 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1621 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1681 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     1741 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     1801 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     1861 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     1921 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     1981 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     2041 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     2101 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     2161 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     2221 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     2281 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     2341 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     2401 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     2461 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     2521 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     2581 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
     2641 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     2701 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     2761 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     2821 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     2881 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     2941 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     3001 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     3061 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3121 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3181 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3241 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     3301 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3361 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3421 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3481 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3541 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3601 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3661 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3721 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3781 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3841 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3901 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     3961 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     4021 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4081 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4141 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     4201 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     4261 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     4321 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     4381 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     4441 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     4501 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     4561 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     4621 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     4681 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     4741 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gccaggaccc
     4801 aacgctgccc gagatctcga tcccgcgaaa t
//
