LOCUS       sequence-389895-        7372 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a N-terminally GFP-TEV-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7372)
  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 7372)
  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: pTS95 pHAGE2 CMVtetO2 GFP-22xGS-TEV-MCS
FEATURES             Location/Qualifiers
     source          1..7372
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    669..794
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1291..1524
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1709..1753
                     /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    2046..2163
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2207..2586
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2587..2790
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2741..2761
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2792..2810
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2813..2831
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2841..2865
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2940..2949
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3711..3731
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     misc_feature    3802..4390
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3855..3875)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4273..4284)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4538..4718
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4765..4783)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4851..4955
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4956..5024,5025..5816)
                     /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(5174..5193)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5987..6575
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6729..6746
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6986..7007
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7022..7027,7028..7045,7046..7052)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7060..7076
                     /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     7065..7087
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7084..7100
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7084..7100
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7150..7169
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(7361..7372,1..622)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 ttcactccca aagaagacaa gatatccttg atctgtggat ctaccacaca caaggctact
       61 tccctgatta gcagaactac acaccagggc caggggtcag atatccactg acctttggat
      121 ggtgctacaa gctagtacca gttgagccag ataaggtaga agaggccaat aaaggagaga
      181 acaccagctt gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtgt
      241 tagagtggag gtttgacagc cgcctagcat ttcatcacgt ggcccgagag ctgcatccgg
      301 agtacttcaa gaactgctga tatcgagctt gctacaaggg actttccgct ggggactttc
      361 cagggaggcg tggcctgggc gggactgggg agtggcgagc cctcagatcc tgcatataag
      421 cagctgcttt ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
      481 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
      541 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
      601 tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac
      661 cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg
      721 ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg
      781 ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt
      841 taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc
      901 tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac
      961 tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata
     1021 cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt
     1081 tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc
     1141 gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg aattatataa
     1201 atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt
     1261 ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc
     1321 agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt
     1381 gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct
     1441 gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag
     1501 atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac
     1561 cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca
     1621 cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt
     1681 aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa
     1741 atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt
     1801 cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt
     1861 gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag
     1921 gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc
     1981 cattcgatta gtgaacggat ctcgacggta tcgccgaatt cacaaatggc agtattcatc
     2041 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     2101 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     2161 tttcgggttt attacaggga cagcagagat ccagtttgga cgcgttgaca ttgattattg
     2221 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     2281 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     2341 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     2401 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     2461 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     2521 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     2581 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     2641 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     2701 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     2761 gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc
     2821 agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat
     2881 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg gctagagcgg
     2941 ccgccatggt gtctaaaggt gaagaacttt ttacgggtgt cgtccccatc ctggttgaat
     3001 tggacggaga cgtgaatggc cataaatttt cagtgagcgg tgaaggagag ggagacgcca
     3061 cttacggtaa acttaccttg aaatttatct gcactaccgg gaaactgccc gtaccctggc
     3121 cgacgctcgt aacaacgctt acctacggcg tgcaatgttt tagcagatac ccagaccata
     3181 tgaagcagca cgattttttc aagagtgcaa tgcctgaggg gtacgtacag gaaaggacga
     3241 tctttttcaa agacgatggt aattataaaa cgcgggctga ggttaaattt gaaggagaca
     3301 cgctcgtgaa tcgaatagag ctgaagggaa tcgacttcaa ggaagacggt aacatcctgg
     3361 gccataaact cgaatataat tacaattcac acaacgtcta tatcatggca gataaacaga
     3421 agaatggcat caaagtcaat tttaagatcc ggcataatat cgaggacgga tcagtacagc
     3481 ttgccgacca ttaccaacag aatacaccga taggagacgg tcccgtcctc ttgccagata
     3541 atcactattt gagcacacaa tccgctcttt ctaaagatcc gaatgaaaag cgcgatcata
     3601 tggtcttgaa agagttcgtc actgccgccg gcataaccgg atctggcagt ggatcaggat
     3661 ctggtagtgg ttctggtagc ggatctggaa gtggcagtgg atctggtagc gagaatttgt
     3721 attttcaggg tggatcttct agtggatccg gttctgctag cggttcaggt tctaccggtg
     3781 gatcatctag ataactcgag taatcaacct ctggattaca aaatttgtga aagattgact
     3841 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3901 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     3961 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     4021 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     4081 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     4141 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtt ggggaaatca
     4201 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     4261 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     4321 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     4381 gcctccccgc ctggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
     4441 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatca
     4501 cctgcaggac aggcgcgccc tgctttttgc ttgtactggg tctctctggt tagaccagat
     4561 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     4621 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     4681 cctcagaccc ttttagtcag tgtggaaaat ctctagcacc cgggcgatta aggaaagggc
     4741 tagatcattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     4801 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     4861 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     4921 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     4981 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5041 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5101 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5161 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     5221 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5281 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5341 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5401 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     5461 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     5521 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     5581 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     5641 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     5701 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     5761 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     5821 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     5881 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     5941 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6001 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6061 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6121 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6181 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6241 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6301 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6361 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6421 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6481 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6541 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6601 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     6661 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     6721 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     6781 tccccgcgcg ttggccgatt cattaatgca gcaagctcat ggctgactaa ttttttttat
     6841 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     6901 ttttggaggc ctaggctttt gcaaaaagct ccccgtggca cgacaggttt cccgactgga
     6961 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     7021 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     7081 acacaggaaa cagctatgac atgattacga atttcacaaa taaagcattt ttttcactgc
     7141 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaactgga
     7201 taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt accactgcca
     7261 attacctgtg gtttcattta ctctaaacct gtgattcctc tgaattattt tcattttaaa
     7321 gaaattgtat ttgttaaata tgtactacaa acttagtagt tggaagggct aa
//
