LOCUS       sequence-389916-        7681 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a C-terminally 3C-SUMOstar-GFP-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7681)
  AUTHORS   Stevens TA, Tomaleri GP, Hazu M, Wei S, Nguyen VN, DeKalb C, 
            Voorhees RM, Pleiner T
  TITLE     A nanobody-based strategy for rapid and scalable purification of 
            human protein complexes.
  JOURNAL   Nat Protoc. 2024 Jan;19(1):127-158. doi: 10.1038/s41596-023-00904-w.
            Epub 2023 Nov 16.
  PUBMED    37974029
REFERENCE   2  (bases 1 to 7681)
  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"; doi: 
            "10.1038/s41596-023-00904-w"; journalName: "Nat Protoc"; date: 
            "2024-01"; volume: "19"; issue: "1"; pages: "127-158"
COMMENT     Sequence Label: pTS108 pHAGE2 CMVtetO2 MCS-3C-SUMOstar-22xGS-GFP
FEATURES             Location/Qualifiers
     source          1..7681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    679..804
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1301..1534
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1719..1763
                     /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    2056..2173
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2217..2596
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2597..2800
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2751..2771
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2802..2820
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2823..2841
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2851..2875
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2950..2959
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3019..3042
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3061..3351
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGG"
     misc_feature    4121..4709
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4174..4194)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4592..4603)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4857..5037
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5084..5102)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5170..5274
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5275..5343,5344..6135)
                     /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(5493..5512)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6306..6894
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7048..7065
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7305..7326
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7341..7346,7347..7364,7365..7371)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7379..7395
                     /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     7384..7406
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7403..7419
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7403..7419
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7469..7488
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(7680..7681,1..632)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 gaagggctaa ttcactccca aagaagacaa gatatccttg atctgtggat ctaccacaca
       61 caaggctact tccctgatta gcagaactac acaccagggc caggggtcag atatccactg
      121 acctttggat ggtgctacaa gctagtacca gttgagccag ataaggtaga agaggccaat
      181 aaaggagaga acaccagctt gttacaccct gtgagcctgc atgggatgga tgacccggag
      241 agagaagtgt tagagtggag gtttgacagc cgcctagcat ttcatcacgt ggcccgagag
      301 ctgcatccgg agtacttcaa gaactgctga tatcgagctt gctacaaggg actttccgct
      361 ggggactttc cagggaggcg tggcctgggc gggactgggg agtggcgagc cctcagatcc
      421 tgcatataag cagctgcttt ttgcctgtac tgggtctctc tggttagacc agatctgagc
      481 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
      541 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
      601 acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg
      661 aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca
      721 agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag
      781 gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa
      841 aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca
      901 agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt
      961 agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca
     1021 ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc
     1081 aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa
     1141 gcggccggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg
     1201 aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa
     1261 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt
     1321 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca
     1381 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc
     1441 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg
     1501 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac
     1561 tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga
     1621 tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa
     1681 tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg
     1741 aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata
     1801 taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac
     1861 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc
     1921 caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca
     1981 gagacagatc cattcgatta gtgaacggat ctcgacggta tcgccgaatt cacaaatggc
     2041 agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag
     2101 aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa
     2161 aattcaaaat tttcgggttt attacaggga cagcagagat ccagtttgga cgcgttgaca
     2221 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     2281 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     2341 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     2401 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     2461 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     2521 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     2581 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     2641 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     2701 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     2761 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga
     2821 tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg
     2881 gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg
     2941 gctagagcgg ccgccatggg atccggttct gctagcggtt caggttctac cggtggatca
     3001 tctagaacta gcggtagctt agaagttttg tttcaaggtc caggtagcgg atctaccgga
     3061 tcggactcag aagtcaatca agaagctaag ccagaggtca agccagaagt caagcctgag
     3121 actcacatca atttaaaggt gtccgatgga tcttcagaaa tcttcttcaa gatcaaaaag
     3181 accactcctt taagaaggct gatggaagcg ttcgctaaaa gacagggtaa ggaaatggac
     3241 tctttaacct tcttgtacga cggtattgag attcaagctg atcagacccc tgaagatttg
     3301 gacatggagg ataacgatat tattgaggct cacagagaac agattggtgg cggatctgga
     3361 agtggatctg gatcaggaag tggttctgga tcaggtagtg gttctggtag tggatcaatg
     3421 gtgtctaaag gtgaagaact ttttacgggt gtcgtcccca tcctggttga attggacgga
     3481 gacgtgaatg gccataaatt ttcagtgagc ggtgaaggag agggagacgc cacttacggt
     3541 aaacttacct tgaaatttat ctgcactacc gggaaactgc ccgtaccctg gccgacgctc
     3601 gtaacaacgc ttacctacgg cgtgcaatgt tttagcagat acccagacca tatgaagcag
     3661 cacgattttt tcaagagtgc aatgcctgag gggtacgtac aggaaaggac gatctttttc
     3721 aaagacgatg gtaattataa aacgcgggct gaggttaaat ttgaaggaga cacgctcgtg
     3781 aatcgaatag agctgaaggg aatcgacttc aaggaagacg gtaacatcct gggccataaa
     3841 ctcgaatata attacaattc acacaacgtc tatatcatgg cagataaaca gaagaatggc
     3901 atcaaagtca attttaagat ccggcataat atcgaggacg gatcagtaca gcttgccgac
     3961 cattaccaac agaatacacc gataggagac ggtcccgtcc tcttgccaga taatcactat
     4021 ttgagcacac aatccgctct ttctaaagat ccgaatgaaa agcgcgatca tatggtcttg
     4081 aaagagttcg tcactgccgc cggcataacc taactcgagt aatcaacctc tggattacaa
     4141 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     4201 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     4261 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     4321 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     4381 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     4441 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     4501 ggtgttgttg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     4561 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     4621 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     4681 tcggatctcc ctttgggccg cctccccgcc tggtaccttt aagaccaatg acttacaagg
     4741 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     4801 cccaacgaag acaagatcac ctgcaggaca ggcgcgccct gctttttgct tgtactgggt
     4861 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     4921 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     4981 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaccc
     5041 gggcgattaa ggaaagggct agatcattct tgaagacgaa agggcctcgt gatacgccta
     5101 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5161 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5221 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5281 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5341 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5401 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5461 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     5521 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5581 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5641 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5701 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5761 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5821 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5881 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5941 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6001 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6061 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6121 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6181 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6241 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6301 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6361 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6421 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6481 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6541 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6601 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6661 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6721 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6781 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6841 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6901 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     6961 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7021 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     7081 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag caagctcatg
     7141 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     7201 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc cccgtggcac
     7261 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     7321 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     7381 gtgagcggat aacaatttca cacaggaaac agctatgaca tgattacgaa tttcacaaat
     7441 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     7501 catgtctgga tcaactggat aactcaagct aaccaaaatc atcccaaact tcccacccca
     7561 taccctatta ccactgccaa ttacctgtgg tttcatttac tctaaacctg tgattcctct
     7621 gaattatttt cattttaaag aaattgtatt tgttaaatat gtactacaaa cttagtagtt
     7681 g
//
