LOCUS       sequence-389900-        7831 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a C-terminally 3C-SUMOstar-ALFA-P2A-BFP-tagged protein in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7831)
  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 7831)
  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: pTS115 pHAGE2 CMVtetO2
            MCS-3C-SUMOstar-22xGS-A...
FEATURES             Location/Qualifiers
     source          1..7831
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             11..644
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    691..816
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1313..1546
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1731..1775
                     /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    2068..2185
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2229..2608
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2609..2812
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2763..2783
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2814..2832
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2835..2853
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2863..2887
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2962..2971
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3031..3054
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3073..3363
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             3433..3471
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             3490..3546
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3574..4275
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    4283..4871
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4336..4356)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4754..4765)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5019..5199
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5246..5264)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5332..5436
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5437..5505,5506..6297)
                     /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(5655..5674)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6468..7056
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7210..7227
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7467..7488
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7503..7508,7509..7526,7527..7533)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7541..7557
                     /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     7546..7568
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7565..7581
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7565..7581
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7631..7650
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 acttagtagt tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg
       61 atctaccaca cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc
      121 agatatccac tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta
      181 gaagaggcca ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg
      241 gatgacccgg agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac
      301 gtggcccgag agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag
      361 ggactttccg ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga
      421 gccctcagat cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga
      481 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
      541 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
      601 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
      661 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
      721 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
      781 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
      841 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
      901 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
      961 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     1021 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     1081 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     1141 accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca
     1201 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1261 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1321 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1381 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1441 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1501 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1561 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1621 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1681 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1741 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1801 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1861 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1921 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     1981 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa
     2041 ttcacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     2101 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     2161 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg
     2221 gacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     2281 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     2341 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     2401 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     2461 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     2521 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     2581 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     2641 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     2701 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     2761 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctccctat
     2821 cagtgataga gatctcccta tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg
     2881 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg
     2941 atccagcctc cggctagagc ggccgccatg ggatccggtt ctgctagcgg ttcaggttct
     3001 accggtggat catctagaac tagcggtagc ttagaagttt tgtttcaagg tccaggtagc
     3061 ggatctaccg gatcggactc agaagtcaat caagaagcta agccagaggt caagccagaa
     3121 gtcaagcctg agactcacat caatttaaag gtgtccgatg gatcttcaga aatcttcttc
     3181 aagatcaaaa agaccactcc tttaagaagg ctgatggaag cgttcgctaa aagacagggt
     3241 aaggaaatgg actctttaac cttcttgtac gacggtattg agattcaagc tgatcagacc
     3301 cctgaagatt tggacatgga ggataacgat attattgagg ctcacagaga acagattggt
     3361 ggcggatctg gaagtggatc tggatcagga agtggttctg gatcaggtag tggttctggt
     3421 agtggatcac catctcgcct ggaagaggaa ctgcgtcgtc gcctgaccga accaaccgga
     3481 ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct
     3541 ggacctggaa cgggcggagc tggttcaacg tctatgagcg agctgattaa ggagaacatg
     3601 cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg cacatccgag
     3661 ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc
     3721 cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag caagaccttc
     3781 atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga gggcttcaca
     3841 tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc
     3901 ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt cacatccaac
     3961 ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgagac gctgtacccc
     4021 gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg cgggagccat
     4081 ctgatcgcaa acatcaagac cacatataga tccaagaaac ccgctaagaa cctcaagatg
     4141 cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa caacgagacc
     4201 tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag caaactgggg
     4261 cacaagctta attaactcga gtaatcaacc tctggattac aaaatttgtg aaagattgac
     4321 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     4381 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     4441 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     4501 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     4561 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     4621 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt tggggaaatc
     4681 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     4741 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     4801 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     4861 cgcctccccg cctggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc
     4921 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc
     4981 acctgcagga caggcgcgcc ctgctttttg cttgtactgg gtctctctgg ttagaccaga
     5041 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     5101 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     5161 ccctcagacc cttttagtca gtgtggaaaa tctctagcac ccgggcgatt aaggaaaggg
     5221 ctagatcatt cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     5281 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     5341 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5401 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5461 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5521 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5581 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5641 acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa
     5701 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5761 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5821 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5881 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5941 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6001 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6061 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6121 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6181 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6241 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6301 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6361 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6421 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6481 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6541 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6601 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6661 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6721 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6781 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6841 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6901 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6961 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7021 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7081 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     7141 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     7201 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     7261 ctccccgcgc gttggccgat tcattaatgc agcaagctca tggctgacta atttttttta
     7321 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     7381 tttttggagg cctaggcttt tgcaaaaagc tccccgtggc acgacaggtt tcccgactgg
     7441 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     7501 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     7561 cacacaggaa acagctatga catgattacg aatttcacaa ataaagcatt tttttcactg
     7621 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcaactgg
     7681 ataactcaag ctaaccaaaa tcatcccaaa cttcccaccc cataccctat taccactgcc
     7741 aattacctgt ggtttcattt actctaaacc tgtgattcct ctgaattatt ttcattttaa
     7801 agaaattgta tttgttaaat atgtactaca a
//
