LOCUS       sequence-389946-        7333 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a N-terminally GFP-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7333)
  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 7333)
  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: pTS93 pHAGE2 CMVtetO2 GFP-22xGS-MCS
FEATURES             Location/Qualifiers
     source          1..7333
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    668..793
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1290..1523
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1708..1752
                     /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    2045..2162
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2206..2585
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2586..2789
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2740..2760
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2791..2809
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2812..2830
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2840..2864
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2939..2948
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    3762..4350
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3815..3835)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4233..4244)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4498..4678
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4725..4743)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4811..4915
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4916..4984,4985..5776)
                     /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(5134..5153)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5947..6535
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6689..6706
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6946..6967
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6982..6987,6988..7005,7006..7012)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7020..7036
                     /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     7025..7047
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7044..7060
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7044..7060
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7110..7129
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(7321..7333,1..621)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 tcactcccaa agaagacaag atatccttga tctgtggatc taccacacac aaggctactt
       61 ccctgattag cagaactaca caccagggcc aggggtcaga tatccactga cctttggatg
      121 gtgctacaag ctagtaccag ttgagccaga taaggtagaa gaggccaata aaggagagaa
      181 caccagcttg ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtgtt
      241 agagtggagg tttgacagcc gcctagcatt tcatcacgtg gcccgagagc tgcatccgga
      301 gtacttcaag aactgctgat atcgagcttg ctacaaggga ctttccgctg gggactttcc
      361 agggaggcgt ggcctgggcg ggactgggga gtggcgagcc ctcagatcct gcatataagc
      421 agctgctttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct
      481 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
      541 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
      601 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
      661 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
      721 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
      781 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
      841 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
      901 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
      961 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     1021 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     1081 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg
     1141 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     1201 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     1261 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     1321 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     1381 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     1441 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     1501 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     1561 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     1621 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     1681 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     1741 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     1801 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     1861 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     1921 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     1981 attcgattag tgaacggatc tcgacggtat cgccgaattc acaaatggca gtattcatcc
     2041 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
     2101 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
     2161 ttcgggttta ttacagggac agcagagatc cagtttggac gcgttgacat tgattattga
     2221 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     2281 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     2341 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     2401 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     2461 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     2521 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     2581 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     2641 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     2701 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     2761 tacggtggga ggtctatata agcagagctc tccctatcag tgatagagat ctccctatca
     2821 gtgatagaga tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc
     2881 cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg ctagagcggc
     2941 cgccatggtg tctaaaggtg aagaactttt tacgggtgtc gtccccatcc tggttgaatt
     3001 ggacggagac gtgaatggcc ataaattttc agtgagcggt gaaggagagg gagacgccac
     3061 ttacggtaaa cttaccttga aatttatctg cactaccggg aaactgcccg taccctggcc
     3121 gacgctcgta acaacgctta cctacggcgt gcaatgtttt agcagatacc cagaccatat
     3181 gaagcagcac gattttttca agagtgcaat gcctgagggg tacgtacagg aaaggacgat
     3241 ctttttcaaa gacgatggta attataaaac gcgggctgag gttaaatttg aaggagacac
     3301 gctcgtgaat cgaatagagc tgaagggaat cgacttcaag gaagacggta acatcctggg
     3361 ccataaactc gaatataatt acaattcaca caacgtctat atcatggcag ataaacagaa
     3421 gaatggcatc aaagtcaatt ttaagatccg gcataatatc gaggacggat cagtacagct
     3481 tgccgaccat taccaacaga atacaccgat aggagacggt cccgtcctct tgccagataa
     3541 tcactatttg agcacacaat ccgctctttc taaagatccg aatgaaaagc gcgatcatat
     3601 ggtcttgaaa gagttcgtca ctgccgccgg cataaccgga tctggcagtg gatcaggatc
     3661 tggtagtggt tctggtagcg gatctggaag tggcagttct agtggatccg gttctgctag
     3721 cggttcaggt tctaccggtg gatcatctag ataactcgag taatcaacct ctggattaca
     3781 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3841 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3901 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3961 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     4021 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     4081 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     4141 tggtgttgtt ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     4201 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     4261 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     4321 gtcggatctc cctttgggcc gcctccccgc ctggtacctt taagaccaat gacttacaag
     4381 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     4441 tcccaacgaa gacaagatca cctgcaggac aggcgcgccc tgctttttgc ttgtactggg
     4501 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     4561 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     4621 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcacc
     4681 cgggcgatta aggaaagggc tagatcattc ttgaagacga aagggcctcg tgatacgcct
     4741 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     4801 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4861 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     4921 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     4981 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5041 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5101 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     5161 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5221 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5281 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5341 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5401 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     5461 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     5521 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     5581 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     5641 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     5701 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5761 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     5821 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5881 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5941 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6001 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6061 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6121 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6181 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6241 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6301 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6361 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6421 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6481 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6541 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6601 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     6661 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     6721 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gcaagctcat
     6781 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     6841 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct ccccgtggca
     6901 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     6961 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     7021 tgtgagcgga taacaatttc acacaggaaa cagctatgac atgattacga atttcacaaa
     7081 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     7141 tcatgtctgg atcaactgga taactcaagc taaccaaaat catcccaaac ttcccacccc
     7201 ataccctatt accactgcca attacctgtg gtttcattta ctctaaacct gtgattcctc
     7261 tgaattattt tcattttaaa gaaattgtat ttgttaaata tgtactacaa acttagtagt
     7321 tggaagggct aat
//
