LOCUS       sequence-326677-        5507 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Gaussia luciferase (GLucM23) targeted to the extracellular surface 
            of the cell membrane and intracellularly labeled with monomeric 
            Venus fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5507)
  AUTHORS   Toth AD, Garger D, Prokop S, Soltesz-Katona E, Varnai P, Balla A, 
            Turu G, Hunyady L
  TITLE     A general method for quantifying ligand binding to unmodified 
            receptors using Gaussia luciferase.
  JOURNAL   J Biol Chem. 2021 Feb 2:100366. doi: 10.1016/j.jbc.2021.100366.
  PUBMED    33545176
REFERENCE   2  (bases 1 to 5507)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol 
            Chem. 2021 Feb 2:100366. doi: 10.1016/j.jbc.2021.100366."
COMMENT     Sequence Label: GLuc-PM-Venus
FEATURES             Location/Qualifiers
     source          1..5507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      255..264
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             261..815
                     /codon_start=1
                     /product="secreted Gaussia luciferase"
                     /label=hGLuc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGEKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIDDIPEIPGFKDLEPIEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGDD"
     CDS             885..1031
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     regulatory      1083..1092
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1089..1091,1092..1094,1095..1808)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1134..1155)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1395..1414)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1742..1763
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1931..2052
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1968..1987)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2022..2041
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2059..2514)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2196..2217)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2408..2427
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2541..2645
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2647..3004
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2647..2838
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2670..2690)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2855..2990
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3039..3833
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3039..3833
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3093..3112)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3703..3722
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4021..4040)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4065..4112
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4441..5029
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4930..4949
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        5204..5507
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 actcagatct cgaggccacc atgggagtca aagttctgtt tgccctgatc tgcatcgctg
      301 tggccgaggc caagcccacc gagaacaacg aagacttcaa catcgtggcc gtggccagca
      361 acttcgcgac cacggatctc gatgctgacc gcgggaagtt gcccggcgag aagctgccgc
      421 tggaggtgct caaagagctt gaagccaatg cccggaaagc tggctgcacc aggggctgtc
      481 tgatctgcct gtcccacatc aagtgcacgc ccaagatgaa gaagttcatc ccaggacgct
      541 gccacaccta cgaaggcgac aaagagtccg cacagggcgg cataggcgag gcgatcgatg
      601 acattcctga gattcctggg ttcaaggact tggagcccat tgagcagttc atcgcacagg
      661 tcgatctgtg tgtggactgc acaactggct gcctcaaagg gcttgccaac gtgcagtgtt
      721 ctgacctgct caagaagtgg ctgccgcaac gctgtgcgac ctttgccagc aagatccagg
      781 gccaggtgga caagatcaag ggggccggtg atgacggagg cggatcaggt ggcggatcca
      841 gatctggtgg cggttctggt ggcggtagtg gtggcggtag taatgctgtg ggccaggaca
      901 cgcaggaggt catcgtggtg ccacactcct tgccctttaa ggtggtggtg atctcagcca
      961 tcctggccct ggtggtgctc accatcatct cccttatcat cctcatcatg ctttggcaga
     1021 agaagccacg taagcttcga attctgcagt cgacggtacc gcgggcccgg gatccaccgg
     1081 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1141 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     1201 ccacctacgg caagctgacc ctgaagctga tctgcaccac cggcaagctg cccgtgccct
     1261 ggcccaccct cgtgaccacc ctgggctacg gcctgcagtg cttcgcccgc taccccgacc
     1321 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     1381 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1441 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1501 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcacc gccgacaagc
     1561 agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac ggcggcgtgc
     1621 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     1681 acaaccacta cctgagctac cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc
     1741 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     1801 acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt
     1861 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     1921 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     1981 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     2041 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     2101 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     2161 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     2221 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     2281 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     2341 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     2401 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     2461 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     2521 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2581 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2641 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     2701 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     2761 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     2821 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     2881 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     2941 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3001 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3061 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3121 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3181 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     3241 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3301 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3361 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3421 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     3481 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     3541 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     3601 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3661 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     3721 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3781 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     3841 tctggggttt gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     3901 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     3961 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     4021 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     4081 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     4141 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     4201 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     4261 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     4321 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4381 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4441 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4501 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4561 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4621 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4681 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4741 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4801 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4861 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4921 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4981 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5041 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5101 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
     5161 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     5221 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     5281 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     5341 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     5401 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     5461 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatg
//
