LOCUS       sequence-246556-        6306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses UBE2J1 with N-terminal Strep-HA tag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6306)
  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 6306)
  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-Strep/HA-UBE2J1
FEATURES             Location/Qualifiers
     source          1..6306
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        80..459
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        460..663
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     614..634
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    665..683
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    686..704
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     714..738
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             909..935
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             966..989
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1026..1049
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     protein_bind    1062..1086
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2041..2065)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2103..2120)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2109..2333
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2466..2485)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2617..2664
                     /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             2672..3694
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2672..3694
                     /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    3824..3945
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3861..3880)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3915..3934
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3994..4010)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3994..4010)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4007..4029)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4018..4034
                     /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(4042..4048,4049..4066,4067..4072))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4087..4108
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4225..4242)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4396..4984)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4476..4495)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5155..5946,5947..6015))
                     /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     5778..5797
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6016..6120)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6195..6214)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
       61 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
      121 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      181 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      241 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      301 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      361 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      421 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      481 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      541 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      601 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      661 gctctcccta tcagtgatag agatctccct atcagtgata gagatcgtcg acgagctcgt
      721 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      781 caccgggacc gatccagcct ccggactcta gcgtttaaac ttaagcttgg taccgagctc
      841 ggatccacta gtccagtgtg gtggaattct gcagatatcc agcacagtgg cggccgctcg
      901 agaccatgta cccatacgat gttcccgact acgccggtac cgagctcgga tccaccatgg
      961 ctagctggag ccacccgcag ttcgagaaag gtggaggttc cggaggtgga tcgggaggtg
     1021 gatcgtggag ccacccgcag ttcgaaaaag cggccgatat cacaagtttg tacaaaaaag
     1081 ttggcatgga gacccgctac aacctgaaga gtccggctgt taaacgttta atgaaagaag
     1141 cggcagaatt gaaagatcca acagatcatt accatgcgca gcctttagag gataaccttt
     1201 ttgaatggca cttcacggtt agagggcccc cagactccga ttttgatgga ggagtttatc
     1261 acgggcggat agtactgcca ccagagtatc ccatgaaacc accaagcatt attctcctaa
     1321 cggctaatgg tcgatttgaa gtgggcaaga aaatctgttt gagcatctca ggccatcatc
     1381 ctgaaacttg gcagccttcg tggagtataa ggacagcatt attagccatc attgggttta
     1441 tgccaacaaa aggagaggga gccataggtt ctctagatta cactcctgag gaaagaagag
     1501 cacttgccaa aaaatcacaa gatttctgtt gtgaaggatg tggctctgcc atgaaggatg
     1561 tcctgttgcc tttaaaatct ggaagcgatt caagccaagc tgaccaagaa gccaaagaac
     1621 tggctaggca aataagcttt aaggcagaag tcaattcatc tggaaagact atctctgagt
     1681 cagacttaaa ccactctttt tcactaactg atttacaaga tgatatacct acaacattcc
     1741 agggtgctac ggccagtaca tcgtacggac tccagaattc ctcagcagca tcctttcatc
     1801 aacctaccca acctgtagct aagaatacct ccatgagccc tcgacagcgc cgggcccagc
     1861 agcagagtca gagaaggttg tctacttcac cagatgtaat ccagggccac cagccaagag
     1921 acaaccacac tgatcatggt gggtcagctg tactgattgt catcctgact ttggcattgg
     1981 cagctcttat attccgacga atatatctgg caaacgaata catatttgac tttgagttat
     2041 aaccaacttt cttgtacaaa gtggtgacgt aagctagggg cccgtttaaa cccgctgatc
     2101 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2161 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2221 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2281 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2341 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2401 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2461 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2521 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2581 caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta
     2641 ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt
     2701 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     2761 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     2821 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
     2881 gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat
     2941 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
     3001 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
     3061 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     3121 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     3181 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     3241 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     3301 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     3361 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     3421 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
     3481 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     3541 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     3601 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     3661 acgccccagc actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac
     3721 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     3781 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     3841 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     3901 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     3961 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4021 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     4081 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4141 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4201 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4261 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4321 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4381 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4441 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4501 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4561 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4621 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4681 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4741 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4801 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4861 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4921 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4981 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5041 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5101 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5161 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5221 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5281 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5341 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5401 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5461 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5521 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5581 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5641 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5701 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5761 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5821 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5881 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5941 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6001 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6061 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6121 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
     6181 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     6241 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     6301 ggcttg
//
