LOCUS       sequence-319281-        5873 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Large subunit of NanoLuc (LgBiT) under CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5873)
  AUTHORS   Somiya M, Kuroda S
  TITLE     Real-Time Luminescence Assay for Cytoplasmic Cargo Delivery of 
            Extracellular Vesicles.
  JOURNAL   Anal Chem. 2021 Mar 24. doi: 10.1021/acs.analchem.1c00339.
  PUBMED    33759512
REFERENCE   2  (bases 1 to 5873)
  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: "Anal Chem. 
            2021 Mar 24. doi: 10.1021/acs.analchem.1c00339."
COMMENT     Sequence Label: pcDNA3.1-LgBiT
FEATURES             Location/Qualifiers
     source          1..5873
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      701..710
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1187..1210
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1230..1247)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1236..1460
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1506..1934
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1593..1612)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1803..1824
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1943..1963)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1948..2277
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2128..2263
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2190..2209
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2344..3138
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2398..2417)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3008..3027
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3312..3433
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3349..3368)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3403..3422
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3482..3498)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3482..3498)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3495..3517)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3506..3522
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3530..3536,3537..3554,3555..3560))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3575..3596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3713..3730)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3884..4472)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3964..3983)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4643..5434,5435..5503))
                     /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     5266..5285
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5504..5608)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5683..5702)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacttaagc ttggtaccga gctcggatcc gccaccatgg tcttcacact
      721 cgaagatttc gttggggact gggaacagac agccgcctac aacctggacc aagtccttga
      781 acagggaggt gtgtccagtt tgctgcagaa tctcgccgtg tccgtaactc cgatccaaag
      841 gattgtccgg agcggtgaaa atgccctgaa gatcgacatc catgtcatca tcccgtatga
      901 aggtctgagc gccgaccaaa tggcccagat cgaagaggtg tttaaggtgg tgtaccctgt
      961 ggatgatcat cactttaagg tgatcctgcc ctatggcaca ctggtaatcg acggggttac
     1021 gccgaacatg ctgaactatt tcggacggcc gtatgaaggc atcgccgtgt tcgacggcaa
     1081 aaagatcact gtaacaggga ccctgtggaa cggcaacaaa attatcgacg agcgcctgat
     1141 cacccccgac ggctccatgc tgttccgagt aaccatcaac agctaagatt acaaggatga
     1201 cgacgataag tgataaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     1261 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1321 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     1381 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     1441 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     1501 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1561 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1621 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1681 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1741 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     1801 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     1861 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     1921 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     1981 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2041 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2101 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2161 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     2221 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     2281 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     2341 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     2401 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     2461 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     2521 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     2581 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     2641 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     2701 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     2761 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     2821 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     2881 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     2941 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     3001 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     3061 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     3121 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     3181 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     3241 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     3301 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3361 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3421 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     3481 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3541 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3601 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3661 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3721 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3781 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3841 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3901 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3961 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4021 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4081 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4141 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4201 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4261 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4321 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4381 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4441 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4501 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4561 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4621 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4681 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4741 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4801 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4861 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4921 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4981 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5041 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5101 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5161 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5221 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5281 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5341 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5401 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5461 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5521 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5581 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     5641 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     5701 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     5761 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     5821 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt
//
