LOCUS       sequence-389909-        7375 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a C-terminally TEV-GFP-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7375)
  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 7375)
  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: pTS105 pHAGE2 CMVtetO2 MCS-TEV-22xGS-GFP
FEATURES             Location/Qualifiers
     source          1..7375
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             2..635
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    682..807
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1304..1537
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1722..1766
                     /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    2059..2176
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2220..2599
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2600..2803
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2754..2774
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2805..2823
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2826..2844
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2854..2878
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2953..2962
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3022..3042
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     misc_feature    3818..4406
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3871..3891)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4289..4300)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4554..4734
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4781..4799)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4867..4971
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4972..5040,5041..5832)
                     /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(5190..5209)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6003..6591
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6745..6762
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7002..7023
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7038..7043,7044..7061,7062..7068)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7076..7092
                     /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     7081..7103
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7100..7116
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7100..7116
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7166..7185
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 ttggaagggc taattcactc ccaaagaaga caagatatcc ttgatctgtg gatctaccac
       61 acacaaggct acttccctga ttagcagaac tacacaccag ggccaggggt cagatatcca
      121 ctgacctttg gatggtgcta caagctagta ccagttgagc cagataaggt agaagaggcc
      181 aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatgggat ggatgacccg
      241 gagagagaag tgttagagtg gaggtttgac agccgcctag catttcatca cgtggcccga
      301 gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc
      361 gctggggact ttccagggag gcgtggcctg ggcgggactg gggagtggcg agccctcaga
      421 tcctgcatat aagcagctgc tttttgcctg tactgggtct ctctggttag accagatctg
      481 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
      541 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
      601 cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa
      661 gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg
      721 gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag
      781 aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg
      841 gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg
      901 gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc
      961 tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga
     1021 tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac
     1081 accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag
     1141 caagcggccg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     1201 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     1261 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     1321 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     1381 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     1441 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     1501 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     1561 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     1621 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     1681 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     1741 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     1801 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     1861 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc
     1921 tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag
     1981 acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgccga attcacaaat
     2041 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga
     2101 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac
     2161 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt ggacgcgttg
     2221 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     2281 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     2341 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     2401 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     2461 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     2521 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     2581 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     2641 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     2701 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     2761 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctcccta tcagtgatag
     2821 agatctccct atcagtgata gagatcgtcg acgagctcgt ttagtgaacc gtcagatcgc
     2881 ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct
     2941 ccggctagag cggccgccat gggatccggt tctgctagcg gttcaggttc taccggtgga
     3001 tcatctagaa ctagcggtag cgagaatttg tattttcagg gtggatctgg atctggaagt
     3061 ggatctggat caggaagtgg ttctggatca ggtagtggtt ctggtagtgg atcaatggtg
     3121 tctaaaggtg aagaactttt tacgggtgtc gtccccatcc tggttgaatt ggacggagac
     3181 gtgaatggcc ataaattttc agtgagcggt gaaggagagg gagacgccac ttacggtaaa
     3241 cttaccttga aatttatctg cactaccggg aaactgcccg taccctggcc gacgctcgta
     3301 acaacgctta cctacggcgt gcaatgtttt agcagatacc cagaccatat gaagcagcac
     3361 gattttttca agagtgcaat gcctgagggg tacgtacagg aaaggacgat ctttttcaaa
     3421 gacgatggta attataaaac gcgggctgag gttaaatttg aaggagacac gctcgtgaat
     3481 cgaatagagc tgaagggaat cgacttcaag gaagacggta acatcctggg ccataaactc
     3541 gaatataatt acaattcaca caacgtctat atcatggcag ataaacagaa gaatggcatc
     3601 aaagtcaatt ttaagatccg gcataatatc gaggacggat cagtacagct tgccgaccat
     3661 taccaacaga atacaccgat aggagacggt cccgtcctct tgccagataa tcactatttg
     3721 agcacacaat ccgctctttc taaagatccg aatgaaaagc gcgatcatat ggtcttgaaa
     3781 gagttcgtca ctgccgccgg cataacctaa ctcgagtaat caacctctgg attacaaaat
     3841 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     3901 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     3961 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     4021 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     4081 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     4141 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     4201 gttgttgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     4261 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     4321 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     4381 gatctccctt tgggccgcct ccccgcctgg tacctttaag accaatgact tacaaggcag
     4441 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     4501 aacgaagaca agatcacctg caggacaggc gcgccctgct ttttgcttgt actgggtctc
     4561 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     4621 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     4681 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcacccggg
     4741 cgattaagga aagggctaga tcattcttga agacgaaagg gcctcgtgat acgcctattt
     4801 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4861 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4921 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4981 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5041 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5101 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5161 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     5221 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5281 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5341 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5401 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5461 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5521 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5581 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5641 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5701 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5761 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5821 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5881 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5941 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6001 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6061 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6121 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6181 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6241 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6301 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6361 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6421 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6481 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6541 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6601 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6661 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     6721 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     6781 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagcaa gctcatggct
     6841 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     6901 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccc gtggcacgac
     6961 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     7021 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     7081 agcggataac aatttcacac aggaaacagc tatgacatga ttacgaattt cacaaataaa
     7141 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     7201 gtctggatca actggataac tcaagctaac caaaatcatc ccaaacttcc caccccatac
     7261 cctattacca ctgccaatta cctgtggttt catttactct aaacctgtga ttcctctgaa
     7321 ttattttcat tttaaagaaa ttgtatttgt taaatatgta ctacaaactt agtag
//
