LOCUS       sequence-257047-        5862 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses UBE2QL1 C88S (catalytically inactive mutant) with 
            C-terminal Strep-HA tag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5862)
  AUTHORS   Koerver L, Papadopoulos C, Liu B, Kravic B, Rota G, Brecht L, 
            Veenendaal T, Polajnar M, Bluemke A, Ehrmann M, Klumperman J, 
            Jaattela M, Behrends C, Meyer H
  TITLE     The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in 
            response to endolysosomal damage.
  JOURNAL   EMBO Rep. 2019 Aug 21:e48014. doi: 10.15252/embr.201948014.
  PUBMED    31432621
REFERENCE   2  (bases 1 to 5862)
  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: "EMBO Rep. 
            2019 Aug 21:e48014. doi: 10.15252/embr.201948014."
COMMENT     Sequence Label: pcDNA5FRT/TO-UBE2QL1-CS-Strep/HA
FEATURES             Location/Qualifiers
     source          1..5862
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        25..404
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        405..608
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     559..579
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    610..628
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    631..649
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     659..683
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    826..850
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1349..1373)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1420..1443
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1480..1503
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1534..1560
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1534..1560
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1604..1621)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1610..1834
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1967..1986)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2118..2165
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2173..3195
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2173..3195
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3325..3446
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3362..3381)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3416..3435
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3495..3511)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3495..3511)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3508..3530)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3519..3535
                     /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(3543..3549,3550..3567,3568..3573))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3588..3609
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3726..3743)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3897..4485)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3977..3996)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4656..5447,5448..5516))
                     /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     5279..5298
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5517..5621)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5696..5715)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
       61 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      121 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      181 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      241 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      301 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      361 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      421 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      481 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      541 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      601 gcagagctct ccctatcagt gatagagatc tccctatcag tgatagagat cgtcgacgag
      661 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata
      721 gaagacaccg ggaccgatcc agcctccgga ctctagcgtt taaacttaag cttggtaccg
      781 agctcggatc cactagtcca gtgtggtgga attctgcaga tatcaacaag tttgtacaaa
      841 aaagcaggct tctcgagcat gaaggagctg caggacatcg cgcgccttag cgaccgcttc
      901 atctccgtgg agctggtgga cgagagcctg ttcgactgga acgtgaagct gcaccaggtg
      961 gacaaggact cggtgctgtg gcaggacatg aaggagacca acaccgagtt catcctgctc
     1021 aacctcacct tccccgacaa cttccccttc tcgccgccct tcatgcgggt gctcagcccg
     1081 cgcctggaga acggctacgt gctggacggc ggcgccatct ccatggagct gctcacgccg
     1141 cgcggctggt ccagcgccta caccgtggag gccgtcatgc gccagttcgc agccagcctg
     1201 gtcaagggcc agggacggat ctgtagaaaa gctggcaaat caaaaaagtc cttcagtcgc
     1261 aaggaagctg aagctacctt taagagtttg gtgaagacgc atgaaaaata tggttgggtc
     1321 accccgcccg tgtccgacgg cggaattcac ccagctttct tgtacaaagt ggttgatatc
     1381 aaggccgccg acatcaccag cctgtacaag aaagtggggt ggagccatcc ccagttcgag
     1441 aagggcggag gatctggcgg cggaagcggc ggaggaagct ggtcccaccc tcagtttgag
     1501 aaaggcaccg agctgggcag caccatggcc agctacccct acgacgtgcc cgactacgcc
     1561 tgatgactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     1621 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     1681 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     1741 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     1801 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     1861 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     1921 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     1981 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2041 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2101 gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga aagtatagga
     2161 acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
     2221 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
     2281 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
     2341 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
     2401 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
     2461 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
     2521 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     2581 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     2641 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     2701 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     2761 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     2821 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     2881 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     2941 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     3001 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     3061 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     3121 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     3181 agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3241 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3301 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     3361 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3421 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     3481 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3541 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3601 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3661 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3721 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3781 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3841 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3901 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3961 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4021 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4081 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4141 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4201 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4261 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4321 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4381 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4441 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4501 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4561 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4621 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4681 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4741 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4801 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4861 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4921 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4981 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5041 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5101 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5161 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5221 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5281 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5341 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5401 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5461 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5521 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5581 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5641 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     5701 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     5761 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     5821 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cg
//
