LOCUS       sequence-389914-        7678 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a C-terminally TEV-SUMOstar-GFP-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7678)
  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 7678)
  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: pTS109 pHAGE2 CMVtetO2 MCS-TEV-SUMOstar-22xGS-GFP
FEATURES             Location/Qualifiers
     source          1..7678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    673..798
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1295..1528
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1713..1757
                     /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    2050..2167
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2211..2590
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2591..2794
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2745..2765
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2796..2814
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2817..2835
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2845..2869
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2944..2953
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3013..3033
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3052..3342
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGG"
     misc_feature    4112..4700
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4165..4185)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4583..4594)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4848..5028
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5075..5093)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5161..5265
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5266..5334,5335..6126)
                     /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(5484..5503)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6297..6885
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7039..7056
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7296..7317
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7332..7337,7338..7355,7356..7362)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7370..7386
                     /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     7375..7397
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7394..7410
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7394..7410
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7460..7479
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(7671..7678,1..626)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 ctaattcact cccaaagaag acaagatatc cttgatctgt ggatctacca cacacaaggc
       61 tacttccctg attagcagaa ctacacacca gggccagggg tcagatatcc actgaccttt
      121 ggatggtgct acaagctagt accagttgag ccagataagg tagaagaggc caataaagga
      181 gagaacacca gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa
      241 gtgttagagt ggaggtttga cagccgccta gcatttcatc acgtggcccg agagctgcat
      301 ccggagtact tcaagaactg ctgatatcga gcttgctaca agggactttc cgctggggac
      361 tttccaggga ggcgtggcct gggcgggact ggggagtggc gagccctcag atcctgcata
      421 taagcagctg ctttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga
      481 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
      541 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
      601 ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg
      661 aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc
      721 gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga
      781 gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt
      841 cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg
      901 gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa
      961 atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat
     1021 aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa
     1081 gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc
     1141 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat
     1201 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa
     1261 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg
     1321 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat
     1381 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc
     1441 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg
     1501 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt
     1561 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga
     1621 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact
     1681 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag
     1741 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat
     1801 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta
     1861 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc
     1921 cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca
     1981 gatccattcg attagtgaac ggatctcgac ggtatcgccg aattcacaaa tggcagtatt
     2041 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     2101 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     2161 aaattttcgg gtttattaca gggacagcag agatccagtt tggacgcgtt gacattgatt
     2221 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     2281 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     2341 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     2401 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     2461 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     2521 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     2581 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     2641 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     2701 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     2761 gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata gagatctccc
     2821 tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg
     2881 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccggctaga
     2941 gcggccgcca tgggatccgg ttctgctagc ggttcaggtt ctaccggtgg atcatctaga
     3001 actagcggta gcgagaattt gtattttcag ggtggtagcg gatctaccgg atcggactca
     3061 gaagtcaatc aagaagctaa gccagaggtc aagccagaag tcaagcctga gactcacatc
     3121 aatttaaagg tgtccgatgg atcttcagaa atcttcttca agatcaaaaa gaccactcct
     3181 ttaagaaggc tgatggaagc gttcgctaaa agacagggta aggaaatgga ctctttaacc
     3241 ttcttgtacg acggtattga gattcaagct gatcagaccc ctgaagattt ggacatggag
     3301 gataacgata ttattgaggc tcacagagaa cagattggtg gcggatctgg aagtggatct
     3361 ggatcaggaa gtggttctgg atcaggtagt ggttctggta gtggatcaat ggtgtctaaa
     3421 ggtgaagaac tttttacggg tgtcgtcccc atcctggttg aattggacgg agacgtgaat
     3481 ggccataaat tttcagtgag cggtgaagga gagggagacg ccacttacgg taaacttacc
     3541 ttgaaattta tctgcactac cgggaaactg cccgtaccct ggccgacgct cgtaacaacg
     3601 cttacctacg gcgtgcaatg ttttagcaga tacccagacc atatgaagca gcacgatttt
     3661 ttcaagagtg caatgcctga ggggtacgta caggaaagga cgatcttttt caaagacgat
     3721 ggtaattata aaacgcgggc tgaggttaaa tttgaaggag acacgctcgt gaatcgaata
     3781 gagctgaagg gaatcgactt caaggaagac ggtaacatcc tgggccataa actcgaatat
     3841 aattacaatt cacacaacgt ctatatcatg gcagataaac agaagaatgg catcaaagtc
     3901 aattttaaga tccggcataa tatcgaggac ggatcagtac agcttgccga ccattaccaa
     3961 cagaatacac cgataggaga cggtcccgtc ctcttgccag ataatcacta tttgagcaca
     4021 caatccgctc tttctaaaga tccgaatgaa aagcgcgatc atatggtctt gaaagagttc
     4081 gtcactgccg ccggcataac ctaactcgag taatcaacct ctggattaca aaatttgtga
     4141 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     4201 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     4261 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     4321 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     4381 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     4441 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtt
     4501 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     4561 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     4621 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     4681 cctttgggcc gcctccccgc ctggtacctt taagaccaat gacttacaag gcagctgtag
     4741 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa
     4801 gacaagatca cctgcaggac aggcgcgccc tgctttttgc ttgtactggg tctctctggt
     4861 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     4921 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     4981 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcacc cgggcgatta
     5041 aggaaagggc tagatcattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     5101 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     5161 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5221 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5281 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5341 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5401 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5461 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     5521 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5581 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5641 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5701 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5761 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5821 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5881 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5941 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6001 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6061 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6121 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6181 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6241 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6301 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6361 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6421 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6481 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6541 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6601 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6661 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6721 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6781 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6841 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6901 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6961 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     7021 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     7081 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gcaagctcat ggctgactaa
     7141 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     7201 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct ccccgtggca cgacaggttt
     7261 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     7321 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     7381 taacaatttc acacaggaaa cagctatgac atgattacga atttcacaaa taaagcattt
     7441 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     7501 atcaactgga taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt
     7561 accactgcca attacctgtg gtttcattta ctctaaacct gtgattcctc tgaattattt
     7621 tcattttaaa gaaattgtat ttgttaaata tgtactacaa acttagtagt tggaaggg
//
