LOCUS       sequence-246551-        6435 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses UBE2QL1 with C-terminal GFP tag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6435)
  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 6435)
  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-GFP
FEATURES             Location/Qualifiers
     source          1..6435
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        97..476
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        477..680
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     631..651
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    682..700
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    703..721
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     731..755
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    898..922
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1421..1445)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1492..1494,1495..1497,1498..2211)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1492..1494,1495..1497,1498..2208)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1537..1558)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1798..1817)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2145..2166
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2249..2266)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2255..2479
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2612..2631)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2763..2810
                     /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             2818..3840
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2818..3840
                     /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    3970..4091
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4007..4026)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4061..4080
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4140..4156)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4140..4156)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4153..4175)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4164..4180
                     /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(4188..4194,4195..4212,4213..4218))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4233..4254
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4371..4388)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4542..5130)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4622..4641)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5301..6092,6093..6161))
                     /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     5924..5943
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6162..6266)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6341..6360)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
       61 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
      121 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      181 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      241 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      301 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      361 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      421 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      481 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      541 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      601 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      661 aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc agtgatagag
      721 atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt
      781 ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagcg tttaaactta
      841 agcttggtac cgagctcgga tccactagtc cagtgtggtg gaattctgca gatatcaaca
      901 agtttgtaca aaaaagcagg cttctcgagc atgaaggagc tgcaggacat cgcgcgcctt
      961 agcgaccgct tcatctccgt ggagctggtg gacgagagcc tgttcgactg gaacgtgaag
     1021 ctgcaccagg tggacaagga ctcggtgctg tggcaggaca tgaaggagac caacaccgag
     1081 ttcatcctgc tcaacctcac cttccccgac aacttcccct tctcgccgcc cttcatgcgg
     1141 gtgctcagcc cgcgcctgga gaacggctac gtgctggacg gcggcgccat ctgcatggag
     1201 ctgctcacgc cgcgcggctg gtccagcgcc tacaccgtgg aggccgtcat gcgccagttc
     1261 gcagccagcc tggtcaaggg ccagggacgg atctgtagaa aagctggcaa atcaaaaaag
     1321 tccttcagtc gcaaggaagc tgaagctacc tttaagagtt tggtgaagac gcatgaaaaa
     1381 tatggttggg tcaccccgcc cgtgtccgac ggcggaattc acccagcttt cttgtacaaa
     1441 gtggttgata tcaaggccgc cgacatcacc agcctgtaca agaaagtggg gatggtgagc
     1501 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1561 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1621 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1681 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1741 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1801 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1861 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1921 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1981 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2041 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2101 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     2161 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtg actcgagtct
     2221 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     2281 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     2341 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     2401 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     2461 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     2521 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2581 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2641 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2701 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc
     2761 tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc cttggccaaa
     2821 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     2881 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     2941 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     3001 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     3061 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     3121 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     3181 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     3241 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     3301 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     3361 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     3421 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     3481 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     3541 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     3601 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     3661 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     3721 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     3781 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag
     3841 cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     3901 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     3961 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4021 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4081 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     4141 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4201 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4261 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4321 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4381 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4441 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4501 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4561 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4621 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4681 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4741 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4801 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4861 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4921 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4981 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5041 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5101 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5161 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5221 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5281 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5341 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5401 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5461 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5521 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5581 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5641 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5701 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5761 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5821 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5881 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5941 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6001 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6061 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6121 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6181 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6241 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     6301 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     6361 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     6421 gcaaaattta agcta
//
