LOCUS       sequence-325326-        7905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HA-Ub noK .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7905)
  AUTHORS   Desai PB, Stuck MW, Lv B, Pazour GJ
  TITLE     Ubiquitin links smoothened to intraflagellar transport to regulate 
            Hedgehog signaling.
  JOURNAL   J Cell Biol. 2020 Jul 6;219(7). pii: 151798. doi: 
            10.1083/jcb.201912104.
  PUBMED    32435793
REFERENCE   2  (bases 1 to 7905)
  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: "J Cell 
            Biol."; date: "2020-07-6"; volume: "219(7). pii: 151798. doi"; 
            pages: " 10.1083/jcb.201912104"
COMMENT     Sequence Label: PD13
FEATURES             Location/Qualifiers
     source          1..7905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2061..2178
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2230..2533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2534..2737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2688..2708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2734..2758
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2826..2852
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2883..3110
                     /codon_start=1
                     /product="human ubiquitin variant in which all of the 
                     lysines have been mutated to arginine"
                     /label=ubiquitin-K0
                     /note="also known as ubiquitin-KO"
                     /translation="MQIFVRTLTGRTITLEVEPSDTIENVRARIQDREGIPPDQQRLIF
                     AGRQLEDGRTLSDYNIQRESTLHLVLRLRGG"
     misc_feature    join(3139..3712,3713..3715,3716..3724)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3305..3322)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3532..3551
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3734..4333
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGSRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3734..3753)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4230..4250
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4347..4935
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4400..4420)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4818..4829)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5083..5263
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5310..5328)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5396..5500
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5501..5569,5570..6361)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5719..5738)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6532..7120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7274..7291
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7531..7552
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7567..7572,7573..7590,7591..7597)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7605..7621
                     /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)."
     primer_bind     7610..7632
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7629..7645
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7629..7645
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7695..7714
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctgcagagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttctcaacaa
     2041 taggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     2101 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     2161 acaaaaattc aaaattttcg ggtttattac tcggacagca gagatccagt ttggactagt
     2221 ggagttccgc gttacataac ttacgcaaaa tggcccgcct ggctgaccgc ccaacgaccc
     2281 ccgcccattg acgtcaataa tgaccaatgt tcccatagta acgccaatag ggactttcca
     2341 ttgacgtcaa tgggtggagt atttacgcaa aactgcccac ttggcagtac atcaagtgta
     2401 tcatttgcca acaacgcccc ctattgacgt caatgacgca aaatggcccg cctggcatta
     2461 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     2521 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     2581 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     2641 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     2701 taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc
     2761 ctggagacgc catccacgct gttttgacct ccattcaaga caccggcggc cttaattaaa
     2821 tgggctaccc ctatgatgtg cctgactacg cagatctcaa tggtggtggt ggtgggtcga
     2881 ccatgcagat cttcgtcaga acgttaaccg gtagaaccat aactctagaa gttgaaccat
     2941 ccgataccat cgaaaacgtt agagctagaa ttcaagacag agaaggcatt ccacctgatc
     3001 aacaaagatt gatctttgcc ggtagacagc tcgaggacgg tagaacgctg tctgattaca
     3061 acattcagag agagtcgacc ttacatcttg tcttaagact aagaggtggt tgagcggccg
     3121 ctaagtaagt aagatccgcc cctctccctc ccccccccta acgttactgg ccgaagccgc
     3181 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt
     3241 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt
     3301 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg
     3361 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca
     3421 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg
     3481 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc
     3541 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct
     3601 gatctggggc ctcggtacac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta
     3661 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac
     3721 aaccatggga accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     3781 cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     3841 cgtcgacccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     3901 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     3961 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggct cgcgcatggc
     4021 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     4081 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg
     4141 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgctggggt
     4201 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     4261 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     4321 gcccggtgcc tgactcgaga gatcctaatc aacctctgga ttacaaaatt tgtgaaagat
     4381 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     4441 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     4501 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     4561 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     4621 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     4681 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     4741 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     4801 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     4861 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     4921 gggccgcctc cccgcctggt acctttaaga ccaatgactt acaaggcagc tgtagatctt
     4981 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     5041 gatcacctgc aggacaggcg cgccctgctt tttgcttgta ctgggtctct ctggttagac
     5101 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     5161 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     5221 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcacccgggc gattaaggaa
     5281 agggctagat cattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     5341 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5401 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5461 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5521 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5581 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5641 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5701 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     5761 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5821 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5881 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5941 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6001 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6061 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6121 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6181 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6241 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6301 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6361 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6421 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6481 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6541 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6601 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6661 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6721 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6781 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6841 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6901 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6961 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7021 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7081 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7141 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     7201 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     7261 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     7321 gcctctcccc gcgcgttggc cgattcatta atgcagcaag ctcatggctg actaattttt
     7381 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     7441 ggcttttttg gaggcctagg cttttgcaaa aagctccccg tggcacgaca ggtttcccga
     7501 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     7561 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     7621 atttcacaca ggaaacagct atgacatgat tacgaatttc acaaataaag catttttttc
     7681 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcaa
     7741 ctggataact caagctaacc aaaatcatcc caaacttccc accccatacc ctattaccac
     7801 tgccaattac ctgtggtttc atttactcta aacctgtgat tcctctgaat tattttcatt
     7861 ttaaagaaat tgtatttgtt aaatatgtac tacaaactta gtagt
//
