LOCUS       sequence-389947-        7378 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a C-terminally 3C-GFP-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7378)
  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 7378)
  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: pTS104 pHAGE2 CMVtetO2 MCS-3C-22xGS-GFP
FEATURES             Location/Qualifiers
     source          1..7378
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             9..642
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    689..814
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1311..1544
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1729..1773
                     /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    2066..2183
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2227..2606
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2607..2810
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2761..2781
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2812..2830
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2833..2851
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2861..2885
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2960..2969
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3029..3052
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     misc_feature    3828..4416
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3881..3901)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4299..4310)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4564..4744
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4791..4809)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4877..4981
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4982..5050,5051..5842)
                     /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(5200..5219)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6013..6601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6755..6772
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7012..7033
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7048..7053,7054..7071,7072..7078)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7086..7102
                     /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     7091..7113
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7110..7126
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7110..7126
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7176..7195
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 ttagtagttg gaagggctaa ttcactccca aagaagacaa gatatccttg atctgtggat
       61 ctaccacaca caaggctact tccctgatta gcagaactac acaccagggc caggggtcag
      121 atatccactg acctttggat ggtgctacaa gctagtacca gttgagccag ataaggtaga
      181 agaggccaat aaaggagaga acaccagctt gttacaccct gtgagcctgc atgggatgga
      241 tgacccggag agagaagtgt tagagtggag gtttgacagc cgcctagcat ttcatcacgt
      301 ggcccgagag ctgcatccgg agtacttcaa gaactgctga tatcgagctt gctacaaggg
      361 actttccgct ggggactttc cagggaggcg tggcctgggc gggactgggg agtggcgagc
      421 cctcagatcc tgcatataag cagctgcttt ttgcctgtac tgggtctctc tggttagacc
      481 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      541 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
      601 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
      661 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
      721 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
      781 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
      841 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
      901 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
      961 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     1021 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     1081 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     1141 cgcacagcaa gcggccggcc gctgatcttc agacctggag gaggagatat gagggacaat
     1201 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     1261 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     1321 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     1381 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     1441 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     1501 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     1561 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     1621 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     1681 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     1741 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     1801 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     1861 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     1921 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     1981 gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta tcgccgaatt
     2041 cacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg
     2101 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac
     2161 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccagtttgga
     2221 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     2281 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     2341 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     2401 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     2461 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     2521 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     2581 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     2641 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     2701 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     2761 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca
     2821 gtgatagaga tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc
     2881 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
     2941 ccagcctccg gctagagcgg ccgccatggg atccggttct gctagcggtt caggttctac
     3001 cggtggatca tctagaacta gcggtagctt agaagttttg tttcaaggtc caggatctgg
     3061 atctggaagt ggatctggat caggaagtgg ttctggatca ggtagtggtt ctggtagtgg
     3121 atcaatggtg tctaaaggtg aagaactttt tacgggtgtc gtccccatcc tggttgaatt
     3181 ggacggagac gtgaatggcc ataaattttc agtgagcggt gaaggagagg gagacgccac
     3241 ttacggtaaa cttaccttga aatttatctg cactaccggg aaactgcccg taccctggcc
     3301 gacgctcgta acaacgctta cctacggcgt gcaatgtttt agcagatacc cagaccatat
     3361 gaagcagcac gattttttca agagtgcaat gcctgagggg tacgtacagg aaaggacgat
     3421 ctttttcaaa gacgatggta attataaaac gcgggctgag gttaaatttg aaggagacac
     3481 gctcgtgaat cgaatagagc tgaagggaat cgacttcaag gaagacggta acatcctggg
     3541 ccataaactc gaatataatt acaattcaca caacgtctat atcatggcag ataaacagaa
     3601 gaatggcatc aaagtcaatt ttaagatccg gcataatatc gaggacggat cagtacagct
     3661 tgccgaccat taccaacaga atacaccgat aggagacggt cccgtcctct tgccagataa
     3721 tcactatttg agcacacaat ccgctctttc taaagatccg aatgaaaagc gcgatcatat
     3781 ggtcttgaaa gagttcgtca ctgccgccgg cataacctaa ctcgagtaat caacctctgg
     3841 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     3901 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     3961 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     4021 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     4081 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     4141 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     4201 attccgtggt gttgttgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
     4261 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     4321 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     4381 agacgagtcg gatctccctt tgggccgcct ccccgcctgg tacctttaag accaatgact
     4441 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     4501 attcactccc aacgaagaca agatcacctg caggacaggc gcgccctgct ttttgcttgt
     4561 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     4621 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     4681 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     4741 agcacccggg cgattaagga aagggctaga tcattcttga agacgaaagg gcctcgtgat
     4801 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     4861 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4921 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4981 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5041 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5101 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5161 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5221 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5281 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5341 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5401 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5461 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5521 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5581 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5641 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5701 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5761 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5821 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5881 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5941 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6001 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6061 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6121 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6181 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6241 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6301 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6361 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6421 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6481 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6541 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6601 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6661 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     6721 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     6781 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagcaa
     6841 gctcatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     6901 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccc
     6961 gtggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     7021 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     7081 tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga ttacgaattt
     7141 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     7201 atcttatcat gtctggatca actggataac tcaagctaac caaaatcatc ccaaacttcc
     7261 caccccatac cctattacca ctgccaatta cctgtggttt catttactct aaacctgtga
     7321 ttcctctgaa ttattttcat tttaaagaaa ttgtatttgt taaatatgta ctacaaac
//
