LOCUS       sequence-495250-        7283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector expressing HA-tagged ubiquitin K63 mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7283)
  AUTHORS   Addgene Research Program
  TITLE     Addgene Ubiquitin Series
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7283)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pLenti Puro HA-Ubiquitin-K63
FEATURES             Location/Qualifiers
     source          1..7283
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     49..69
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        51..69
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        97..323
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             324..504
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    551..676
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1169..1402
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1587..1631
                     /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    1929..2046
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2139..2342
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2293..2313
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2390..2416
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2426..2653
                     /codon_start=1
                     /product="human ubiquitin variant containing Lys-63, with 
                     all other lysines mutated to arginine"
                     /label=ubiquitin-K63
                     /translation="MQIFVRTLTGRTITLEVEPSDTIENVRARIQDREGIPPDQQRLIF
                     AGRQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             2663..2719
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2720..3319
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2720..2739)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3216..3236
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3335..3923
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3388..3408)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3806..3817)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3995..4228
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4300..4421
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4337..4356)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4391..4410
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4461..4596
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4616..4635)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4617..4635)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4667..4684)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4667..4683)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4676..4698)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4892..4911)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5011..5033
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5071..5089)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5157..5261
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5262..5330,5331..6122)
                     /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     complement(5480..5499)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6293..6881
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6782..6801
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7035..7052
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7169..7190
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7205..7210,7211..7228,7229..7235)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7243..7259
                     /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     7248..7270
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7267..7283
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7267..7283
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 catgattacg ccaagcttgc atgcctgcag catgattacg ccaagcgcgc aattaaccct
       61 cactaaaggg aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt
      121 agtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc
      181 gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga
      241 cgggtctgac atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta
      301 agtgcctagc tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc
      361 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
      421 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
      481 agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa
      541 accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga
      601 ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga
      661 tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg
      721 gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga
      781 gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat
      841 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
      901 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
      961 tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc
     1021 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
     1081 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
     1141 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
     1201 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
     1261 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
     1321 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
     1381 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
     1441 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
     1501 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
     1561 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat
     1621 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
     1681 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga
     1741 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
     1801 gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca
     1861 ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc
     1921 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     1981 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     2041 aattttcggg tttattacag ggacagcaga gatccagttt ggttaattag ctagctaggt
     2101 cttgaaagga gtgggaattg gctccggtgc ccgtcagtgt gatgcggttt tggcagtaca
     2161 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     2221 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     2281 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     2341 ctggtttagt gaaccgtcag atccgctagc gctaccggtc gccaccatgt acccatacga
     2401 tgttccggat tacgctagcg gatccatgca gatcttcgtc agaacgttaa ccggtagaac
     2461 cataactcta gaagttgaac catccgatac catcgaaaac gttagagcta gaattcaaga
     2521 cagagaaggc attccacctg atcaacaaag attgatcttt gccggtagac agctcgagga
     2581 cggtagaacg ctgtctgatt acaacattca gaaggagtcc accctgcacc tggtcctccg
     2641 tctcagaggt ggtggatccg gcgcaacaaa cttctctctg ctgaaacaag ccggagatgt
     2701 cgaagagaat cctggaccga tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     2761 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     2821 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     2881 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     2941 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     3001 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     3061 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca
     3121 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     3181 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     3241 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     3301 ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa cctctggatt acaaaatttg
     3361 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     3421 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     3481 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     3541 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     3601 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     3661 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     3721 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
     3781 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     3841 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     3901 ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta
     3961 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     4021 agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg
     4081 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     4141 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     4201 ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat
     4261 ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat
     4321 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4381 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta
     4441 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     4501 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     4561 tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta
     4621 tagtgagtcg tattacgcgc gctccccggg taccgagctc gaattcactg gccgtcgttt
     4681 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     4741 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     4801 tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg
     4861 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4921 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4981 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     5041 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     5101 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     5161 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5221 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5281 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5341 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5401 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5461 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5521 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5581 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5641 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5701 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5761 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5821 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5881 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5941 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6001 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6061 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6121 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6181 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6241 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6301 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6361 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6421 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6481 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6541 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6601 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6661 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6721 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6781 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6841 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6901 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     6961 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     7021 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     7081 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     7141 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     7201 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     7261 ttcacacagg aaacagctat gac
//
