LOCUS       sequence-456690-        7527 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Firefly luciferase in fusion with a fragment of human Skp1 protein 
            through a flexible linker and a TEV viral protease cleavage site. 
            Expression of fLuc protein is deinhibited by TEV site cleavage..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7527)
  AUTHORS   Verbic A, Lebar T, Praznik A, Jerala R
  TITLE     Subunits of an E3 Ligase Complex as Degrons for Efficient 
            Degradation of Cytosolic, Nuclear, and Membrane Proteins.
  JOURNAL   ACS Synth Biol. 2024 Mar 15;13(3):792-803. doi: 
            10.1021/acssynbio.3c00588. Epub 2024 Feb 26.
  PUBMED    38404221
REFERENCE   2  (bases 1 to 7527)
  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"; doi: 
            "10.1021/acssynbio.3c00588"; journalName: "ACS Synth Biol"; date: 
            "2024-03-15- 15"; volume: "13"; issue: "3"; pages: "792-803"
COMMENT     Sequence Label: pcDNA3.1-myc:Skp1(dC111):gs6:TEVs:gs6:fLuc:HA
FEATURES             Location/Qualifiers
     source          1..7527
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1223..1249
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(1268..1286)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1269..1286)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1306..1323)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1312..1536
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1582..2010
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1669..1688)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1879..1900
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2019..2039)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2024..2353
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2204..2339
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2266..2285
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2420..3214
                     /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(2474..2493)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3084..3103
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3388..3509
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3425..3444)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3479..3498
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3558..3574)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3558..3574)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3571..3593)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3582..3598
                     /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(3606..3612,3613..3630,3631..3636))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3651..3672
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3789..3806)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3960..4548)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4040..4059)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4719..5510,5511..5579))
                     /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     5342..5361
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5580..5684)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5759..5778)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5950..6329
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6330..6533
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6484..6504
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6578..6597
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6578..6596
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6659..6668
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6668..6697
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             7049..7069
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(7088..7527,1..1207)
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="EDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH
                     IEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA
                     NDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM
                     YTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR
                     DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKI
                     QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG
                     LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG
                     YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL
                     QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF
                     VDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
ORIGIN
        1 gatcatcatc atggatagca agaccgacta ccagggcttc caaagcatgt acaccttcgt
       61 gacttcccat ttgccacccg gcttcaacga gtacgacttc gtgcccgaga gcttcgaccg
      121 ggacaaaacc atcgccctga tcatgaacag tagtggcagt accggattgc ccaagggcgt
      181 agccctaccg caccgcaccg cttgtgtccg attcagtcat gcccgcgacc ccatcttcgg
      241 caaccagatc atccccgaca ccgctatcct cagcgtggtg ccatttcacc acggcttcgg
      301 catgttcacc acgctgggct acttgatctg cggctttcgg gtcgtgctca tgtaccgctt
      361 cgaggaggag ctattcttgc gcagcttgca agactataag attcaatctg ccctgctggt
      421 gcccacacta tttagcttct tcgctaagag cactctcatc gacaagtacg acctaagcaa
      481 cttgcacgag atcgccagcg gcggggcgcc gctcagcaag gaggtaggtg aggccgtggc
      541 caaacgcttc cacctaccag gcatccgcca gggctacggc ctgacagaaa caaccagcgc
      601 cattctgatc acccccgaag gggacgacaa gcctggcgca gtaggcaagg tggtgccctt
      661 cttcgaggct aaggtggtgg acttggacac cggtaagaca ctgggtgtga accagcgcgg
      721 cgagctgtgc gtccgtggcc ccatgatcat gagcggctac gttaacaacc ccgaggctac
      781 aaacgctctc atcgacaagg acggctggct gcacagcggc gacatcgcct actgggacga
      841 ggacgagcac ttcttcatcg tggaccggct gaagagcctg atcaaataca agggctacca
      901 ggtagcccca gccgaactgg agagcatcct gctgcaacac cccaacatct tcgacgccgg
      961 ggtcgccggc ctgcccgacg acgatgccgg cgagctgccc gccgcagtcg tcgtgctgga
     1021 acacggtaaa accatgaccg agaaggagat cgtggactat gtggccagcc aggttacaac
     1081 cgccaagaag ctgcgcggtg gtgttgtgtt cgtggacgag gtgcctaaag gactgaccgg
     1141 caagttggac gcccgcaaga tccgcgagat tctcattaag gccaagaagg gcggcaagat
     1201 cgccgtgaat agtggttctg gatacccata cgatgttcca gattacgctt aatctagagg
     1261 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt
     1321 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     1381 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     1441 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     1501 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     1561 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1621 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1681 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1741 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1801 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1861 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1921 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1981 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     2041 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2101 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2161 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     2221 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     2281 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     2341 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     2401 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     2461 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     2521 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     2581 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     2641 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     2701 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     2761 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     2821 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     2881 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     2941 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     3001 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     3061 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     3121 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     3181 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     3241 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     3301 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     3361 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     3421 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     3481 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     3541 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3601 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3661 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3721 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     3781 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3841 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3901 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3961 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4021 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4081 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4141 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4201 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4261 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4321 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4381 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4441 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4501 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4561 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4621 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4681 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4741 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4801 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4861 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4921 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4981 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5041 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5101 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5161 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5221 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5281 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5341 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5401 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5461 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5521 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5581 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5641 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5701 gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta
     5761 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     5821 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     5881 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     5941 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     6001 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     6061 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     6121 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     6181 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     6241 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     6301 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     6361 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     6421 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     6481 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc
     6541 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga
     6601 cccaagcttg gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattcgc
     6661 caccatggag cagaagctga tcagcgagga ggacctgccc agcatcaagc tgcagagcag
     6721 cgacggcgag atcttcgagg tggacgtgga gatcgccaag cagagcgtga ccatcaagac
     6781 catgctggag gacctgggca tggacgacga gggcgacgac gaccccgtgc ccctgcccaa
     6841 cgtgaacgcc gccatcctga agaaggtgat ccagtggtgc acccaccaca aggacgaccc
     6901 tcctcctcct gaggacgacg agaacaagga gaagaggacc gacgacatcc ccgtgtggga
     6961 ccaggagttc ctgaaggtgg accagggcac cctgttcgag ctgatcctgg ccgccaacta
     7021 cctggacatc ggcggctctg gcggcggcga aaatttgtat ttccagagtg gcggctccgg
     7081 aggctctgaa gatgccaaaa acattaagaa gggcccagcg ccattctacc cactcgaaga
     7141 cgggaccgcc ggcgagcagc tgcacaaagc catgaagcgc tacgccctgg tgcccggcac
     7201 catcgccttt accgacgcac atatcgaggt ggacattacc tacgccgagt acttcgagat
     7261 gagcgttcgg ctggcagaag ctatgaagcg ctatgggctg aatacaaacc atcggatcgt
     7321 ggtgtgcagc gagaatagct tgcagttctt catgcccgtg ttgggtgccc tgttcatcgg
     7381 tgtggctgtg gccccagcta acgacatcta caacgagcgc gagctgctga acagcatggg
     7441 catcagccag cccaccgtcg tattcgtgag caagaaaggg ctgcaaaaga tcctcaacgt
     7501 gcaaaagaag ctaccgatca tacaaaa
//
