LOCUS       sequence-336168-        5992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing KB-1753-Nluc I9A/W10A in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5992)
  AUTHORS   Maziarz M, Park JC, Leyme A, Marivin A, Garcia-Lopez A, Patel PP, 
            Garcia-Marcos M
  TITLE     Revealing the Activity of Trimeric G-proteins in Live Cells with a 
            Versatile Biosensor Design.
  JOURNAL   Cell. 2020 Jun 27. pii: S0092-8674(20)30752-2. doi: 
            10.1016/j.cell.2020.06.020.
  PUBMED    32634377
REFERENCE   2  (bases 1 to 5992)
  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: "Cell. 2020 
            Jun 27. pii: S0092-8674(20)30752-2. doi: 
            10.1016/j.cell.2020.06.020."
COMMENT     Sequence Label: pcDNA3.1-mas-KB-1753-Nluc I9A/W10A
FEATURES             Location/Qualifiers
     source          1..5992
                     /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"
     CDS             670..690
                     /codon_start=1
                     /product="N-myristoylation signal from Src kinase (Pellman 
                     et al., 1985; Kaplan et al., 1988)"
                     /label=myr
                     /translation="MGSSKSK"
     CDS             796..1311
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(1349..1366)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1355..1579
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1625..2053
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1712..1731)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1922..1943
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2062..2082)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2067..2396
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2247..2382
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2309..2328
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2463..3257
                     /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(2517..2536)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3127..3146
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3431..3552
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3468..3487)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3522..3541
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3601..3617)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3601..3617)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3614..3636)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3625..3641
                     /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(3649..3655,3656..3673,3674..3679))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3694..3715
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3832..3849)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4003..4591)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4083..4102)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4762..5553,5554..5622))
                     /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     5385..5404
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5623..5727)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5802..5821)
                     /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 gctagcacca tggggagtag caagagcaag acgtcgaatt ccggaatcaa gcttggtgga
      721 tcttctcgag gttactacca tggtgctgcg gtgggtgaag aaggtcgact ttctcgaggt
      781 ggcaccggtg gatccatggt cttcacactc gaagatttcg ttggggactg gcgacagaca
      841 gccggctaca acctggacca agtccttgaa cagggaggtg tgtccagttt gtttcagaat
      901 ctcggggtgt ccgtaactcc gatccaaagg attgtcctga gcggtgaaaa tgggctgaag
      961 atcgacatcc atgtcatcat cccgtatgaa ggtctgagcg gcgaccaaat gggccagatc
     1021 gaaaaaattt ttaaggtggt gtaccctgtg gatgatcatc actttaaggt gatcctgcac
     1081 tatggcacac tggtaatcga cggggttacg ccgaacatga tcgactattt cggacggccg
     1141 tatgaaggca tcgccgtgtt cgacggcaaa aagatcactg taacagggac cctgtggaac
     1201 ggcaacaaaa ttatcgacga gcgcctgatc aaccccgacg gctccctgct gttccgagta
     1261 accatcaacg gagtgaccgg ctggcggctg tgcgaacgca ttctggcgta actcgagtct
     1321 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     1381 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1441 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     1501 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     1561 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     1621 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1681 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     1741 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     1801 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     1861 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     1921 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     1981 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2041 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2101 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2161 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2221 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     2281 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     2341 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     2401 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     2461 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     2521 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     2581 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     2641 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     2701 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     2761 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     2821 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     2881 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     2941 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     3001 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3061 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3121 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     3181 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     3241 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     3301 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     3361 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     3421 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3481 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3541 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     3601 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3661 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     3721 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     3781 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     3841 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3901 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3961 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4021 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4081 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4141 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4201 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4261 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4321 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4381 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4441 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4501 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4561 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4621 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4681 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4741 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4801 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4861 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4921 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4981 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5041 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5101 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5161 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5221 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5281 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5341 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5401 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5461 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5521 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5581 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5641 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5701 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     5761 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     5821 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     5881 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     5941 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg tt
//
