LOCUS       Exported                6253 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses FLAG-OTUB1 WT in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    FLAG-OTUB1 WT-pcDNA3.1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6253)
  AUTHORS   Pasupala N, Morrow ME, Que LT, Malynn BA, Ma A, Wolberger C
  TITLE     OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating 
            enzyme UBE2E1 by preventing its autoubiquitination.
  JOURNAL   J Biol Chem. 2018 Nov 23;293(47):18285-18295. doi: 
            10.1074/jbc.RA118.004677. Epub 2018 Oct 3.
  PUBMED    30282802
REFERENCE   2  (bases 1 to 6253)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6253
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             686..751
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1610..1627)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1616..1840
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1886..2314
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1973..1992)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2183..2204
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2323..2343)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2328..2657
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2508..2643
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2570..2589
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2724..3518
                     /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(2778..2797)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3388..3407
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3692..3813
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3729..3748)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3783..3802
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3862..3878)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3862..3878)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3875..3897)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3886..3902
                     /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(3910..3940)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3955..3976
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4093..4110)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4264..4852)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4344..4363)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5023..5883)
                     /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     5646..5665
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5884..5988)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6063..6082)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacttaagc ttatggacta caaagaccat gacggtgatt ataaagatca
      721 tgacatcgat tacaaggatg acgatgacaa gaattcaatg gcggcggagg aacctcagca
      781 gcagaagcag gagccgctgg gcagcgactc cgaaggtgtt aactgtctgg cctatgatga
      841 agccatcatg gctcagcagg accgaattca gcaagagatt gctgtgcaga accctctggt
      901 gtcagagcgg ctggagctct cggtcctata caaggagtat gctgaagatg acaacatcta
      961 tcaacagaag atcaaggacc tccacaaaaa gtactcgtac atccgcaaga ccaggcctga
     1021 cggcaactgt ttctatcggg ctttcggatt ctcccacttg gaggcactgc tggatgacag
     1081 caaggagttg cagcggttca aggctgtgtc tgccaagagc aaggaagacc tggtgtccca
     1141 gggcttcact gaattcacaa ttgaggattt ccacaacacg ttcatggacc tgattgagca
     1201 ggtggagaag cagacctctg tcgccgacct gctggcctcc ttcaatgacc agagcacctc
     1261 cgactacctt gtggtctacc tgcggctgct cacctcgggc tacctgcagc gcgagagcaa
     1321 gttcttcgag cacttcatcg agggtggacg gactgtcaag gagttctgcc agcaggaggt
     1381 ggagcccatg tgcaaggaga gcgaccacat ccacatcatt gcgctggccc aggccctcag
     1441 cgtgtccatc caggtggagt acatggaccg cggcgagggc ggcaccacca atccgcacat
     1501 cttccctgag ggctccgagc ccaaggtcta ccttctctac cggcctggac actacgatat
     1561 cctctacaaa tagtcgactc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg
     1621 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1681 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1741 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     1801 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     1861 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     1921 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     1981 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2041 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2101 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2161 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2221 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2281 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     2341 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2401 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2461 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2521 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2581 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2641 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     2701 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     2761 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     2821 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     2881 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     2941 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3001 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3061 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3121 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3181 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3241 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3301 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3361 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3421 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3481 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     3541 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     3601 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3661 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     3721 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3781 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     3841 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3901 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3961 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4021 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4081 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4141 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4201 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4261 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4321 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4381 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4441 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4501 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4561 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4621 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4681 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4741 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4801 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4861 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4921 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4981 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5041 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5101 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5161 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5221 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5281 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5341 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5401 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5461 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5521 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5581 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5641 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5701 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5761 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5821 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5881 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5941 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6001 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     6061 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     6121 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     6181 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     6241 agatatacgc gtt
//
