LOCUS       sequence-389896-        7636 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a N-terminally GFP-SUMOstar-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7636)
  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 7636)
  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: pTS97 pHAGE2 CMVtetO2 GFP-22xGS-SUMOstar-MCS
FEATURES             Location/Qualifiers
     source          1..7636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2219..2598
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2599..2802
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2753..2773
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2804..2822
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2825..2843
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2853..2877
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2952..2961
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3723..4013
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGG"
     misc_feature    4078..4666
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4131..4151)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4549..4560)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4814..4994
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5041..5059)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5127..5231
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5232..5300,5301..6092)
                     /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(5450..5469)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6263..6851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7005..7022
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7262..7283
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7298..7303,7304..7321,7322..7328)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7336..7352
                     /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     7341..7363
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7360..7376
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7360..7376
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7426..7445
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gacgcgttga
     2221 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     2281 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     2341 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     2401 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     2461 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     2521 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     2581 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     2641 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     2701 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     2761 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctccctat cagtgataga
     2821 gatctcccta tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg tcagatcgcc
     2881 tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc
     2941 cggctagagc ggccgccatg gtgtctaaag gtgaagaact ttttacgggt gtcgtcccca
     3001 tcctggttga attggacgga gacgtgaatg gccataaatt ttcagtgagc ggtgaaggag
     3061 agggagacgc cacttacggt aaacttacct tgaaatttat ctgcactacc gggaaactgc
     3121 ccgtaccctg gccgacgctc gtaacaacgc ttacctacgg cgtgcaatgt tttagcagat
     3181 acccagacca tatgaagcag cacgattttt tcaagagtgc aatgcctgag gggtacgtac
     3241 aggaaaggac gatctttttc aaagacgatg gtaattataa aacgcgggct gaggttaaat
     3301 ttgaaggaga cacgctcgtg aatcgaatag agctgaaggg aatcgacttc aaggaagacg
     3361 gtaacatcct gggccataaa ctcgaatata attacaattc acacaacgtc tatatcatgg
     3421 cagataaaca gaagaatggc atcaaagtca attttaagat ccggcataat atcgaggacg
     3481 gatcagtaca gcttgccgac cattaccaac agaatacacc gataggagac ggtcccgtcc
     3541 tcttgccaga taatcactat ttgagcacac aatccgctct ttctaaagat ccgaatgaaa
     3601 agcgcgatca tatggtcttg aaagagttcg tcactgccgc cggcataacc ggatctggca
     3661 gtggatcagg atctggtagt ggttctggta gcggatctgg aagtggcagt ggatctaccg
     3721 gatcggactc agaagtcaat caagaagcta agccagaggt caagccagaa gtcaagcctg
     3781 agactcacat caatttaaag gtgtccgatg gatcttcaga aatcttcttc aagatcaaaa
     3841 agaccactcc tttaagaagg ctgatggaag cgttcgctaa aagacagggt aaggaaatgg
     3901 actctttaac cttcttgtac gacggtattg agattcaagc tgatcagacc cctgaagatt
     3961 tggacatgga ggataacgat attattgagg ctcacagaga acagattggt ggctctagtg
     4021 gatccggttc tgctagcggt tcaggttcta ccggtggatc atctagataa ctcgagtaat
     4081 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     4141 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     4201 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     4261 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     4321 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     4381 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     4441 ggcactgaca attccgtggt gttgttgggg aaatcatcgt cctttccttg gctgctcgcc
     4501 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     4561 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     4621 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctgg tacctttaag
     4681 accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact
     4741 ggaagggcta attcactccc aacgaagaca agatcacctg caggacaggc gcgccctgct
     4801 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     4861 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     4921 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     4981 gaaaatctct agcacccggg cgattaagga aagggctaga tcattcttga agacgaaagg
     5041 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     5101 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5161 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5221 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5281 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5341 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5401 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5461 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5521 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5581 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5641 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5701 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     5761 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     5821 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5881 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5941 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6001 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6061 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6121 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6181 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6241 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6301 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6361 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6421 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6481 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6541 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6601 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6661 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6721 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6781 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6841 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6901 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6961 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     7021 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     7081 aatgcagcaa gctcatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     7141 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     7201 aaagctcccc gtggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     7261 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     7321 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga
     7381 ttacgaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     7441 tcatcaatgt atcttatcat gtctggatca actggataac tcaagctaac caaaatcatc
     7501 ccaaacttcc caccccatac cctattacca ctgccaatta cctgtggttt catttactct
     7561 aaacctgtga ttcctctgaa ttattttcat tttaaagaaa ttgtatttgt taaatatgta
     7621 ctacaaactt agtagt
//
