LOCUS       sequence-325309-        7744 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Flag-Ub noK .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7744)
  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 7744)
  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: PD19
FEATURES             Location/Qualifiers
     source          1..7744
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(41..61)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(459..470)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             724..904
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(951..969)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1037..1141
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1142..1210,1211..2002)
                     /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(1360..1379)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2173..2761
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2915..2932
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3172..3193
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3208..3213,3214..3231,3232..3238)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3246..3262
                     /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     3251..3273
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3270..3286
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3270..3286
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3336..3355
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             3547..4180
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    4227..4352
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4849..5082
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5267..5311
                     /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    5607..5724
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        5776..6079
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6080..6283
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6234..6254
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6280..6304
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             6385..6450
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6484..6711
                     /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(6734..7307,7308..7310,7311..7319)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(6900..6917)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     7127..7146
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             7320..7718
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    join(7732..7744,1..576)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
ORIGIN
        1 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
       61 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
      121 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
      181 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
      241 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
      301 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
      361 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
      421 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
      481 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
      541 agacgagtcg gatctccctt tgggccgcct ccccgcctgg tacctttaag accaatgact
      601 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
      661 attcactccc aacgaagaca agatcacctg caggacaggc gcgccctgct ttttgcttgt
      721 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      781 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      841 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
      901 agcacccggg cgattaagga aagggctaga tcattcttga agacgaaagg gcctcgtgat
      961 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     1021 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1081 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     1141 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     1201 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     1261 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     1321 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     1381 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     1441 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     1501 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     1561 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     1621 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     1681 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     1741 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     1801 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     1861 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     1921 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     1981 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     2041 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     2101 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     2161 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2221 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2281 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     2341 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2401 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2461 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     2521 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     2581 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     2641 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2701 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     2761 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     2821 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     2881 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     2941 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagcaa
     3001 gctcatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     3061 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccc
     3121 gtggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     3181 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     3241 tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga ttacgaattt
     3301 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     3361 atcttatcat gtctggatca actggataac tcaagctaac caaaatcatc ccaaacttcc
     3421 caccccatac cctattacca ctgccaatta cctgtggttt catttactct aaacctgtga
     3481 ttcctctgaa ttattttcat tttaaagaaa ttgtatttgt taaatatgta ctacaaactt
     3541 agtagttgga agggctaatt cactcccaaa gaagacaaga tatccttgat ctgtggatct
     3601 accacacaca aggctacttc cctgattagc agaactacac accagggcca ggggtcagat
     3661 atccactgac ctttggatgg tgctacaagc tagtaccagt tgagccagat aaggtagaag
     3721 aggccaataa aggagagaac accagcttgt tacaccctgt gagcctgcat gggatggatg
     3781 acccggagag agaagtgtta gagtggaggt ttgacagccg cctagcattt catcacgtgg
     3841 cccgagagct gcatccggag tacttcaaga actgctgata tcgagcttgc tacaagggac
     3901 tttccgctgg ggactttcca gggaggcgtg gcctgggcgg gactggggag tggcgagccc
     3961 tcagatcctg catataagca gctgcttttt gcctgtactg ggtctctctg gttagaccag
     4021 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     4081 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     4141 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
     4201 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
     4261 gcacggcaag aggcgagggg cggcgactgc agagtacgcc aaaaattttg actagcggag
     4321 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
     4381 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
     4441 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
     4501 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
     4561 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
     4621 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
     4681 cacagcaagc ggccggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     4741 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     4801 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     4861 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     4921 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     4981 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     5041 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     5101 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     5161 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     5221 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     5281 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     5341 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     5401 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     5461 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
     5521 gagagacaga gacagatcca ttcgattagt gaacggatct cgacggtatc gccgaattct
     5581 caacaatagg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     5641 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     5701 caaattacaa aaattcaaaa ttttcgggtt tattactcgg acagcagaga tccagtttgg
     5761 actagtggag ttccgcgtta cataacttac gcaaaatggc ccgcctggct gaccgcccaa
     5821 cgacccccgc ccattgacgt caataatgac caatgttccc atagtaacgc caatagggac
     5881 tttccattga cgtcaatggg tggagtattt acgcaaaact gcccacttgg cagtacatca
     5941 agtgtatcat ttgccaacaa cgccccctat tgacgtcaat gacgcaaaat ggcccgcctg
     6001 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     6061 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     6121 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     6181 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     6241 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca
     6301 gatcgcctgg agacgccatc cacgctgttt tgacctccat tcaagacacc ggcggcctta
     6361 attaatgatc aggatcccac catggactac aaagaccatg acggtgatta taaagatcat
     6421 gacatcgatt acaaggatga cgatgacaag gcagatctca atggtggtgg tggtgggtcg
     6481 accatgcaga tcttcgtcag aacgttaacc ggtagaacca taactctaga agttgaacca
     6541 tccgatacca tcgaaaacgt tagagctaga attcaagaca gagaaggcat tccacctgat
     6601 caacaaagat tgatctttgc cggtagacag ctcgaggacg gtagaacgct gtctgattac
     6661 aacattcaga gagagtcgac cttacatctt gtcttaagac taagaggtgg ttgagcttag
     6721 taagtaagat ccgcccctct ccctcccccc ccctaacgtt actggccgaa gccgcttgga
     6781 ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa
     6841 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc
     6901 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc
     6961 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg
     7021 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
     7081 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
     7141 cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct
     7201 ggggcctcgg tacacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc
     7261 cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataatatg gccacaacca
     7321 tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg gctacaatca
     7381 acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct agcgacggcc
     7441 gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca gaactcgtgg
     7501 tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa
     7561 atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt ctcgatctgc
     7621 atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca gttgggattc
     7681 gtgaattgct gccctctggt tatgtgtggg agggctaact cgagagatcc taatcaacct
     7741 ctgg
//
