LOCUS       sequence-389901-        7828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a C-terminally TEV-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 7828)
  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 7828)
  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: pTS116 pHAGE2 CMVtetO2
            MCS-TEV-SUMOstar-22xGS-...
FEATURES             Location/Qualifiers
     source          1..7828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             10..643
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    690..815
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1312..1545
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1730..1774
                     /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    2067..2184
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2228..2607
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2608..2811
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2762..2782
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2813..2831
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2834..2852
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2862..2886
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2961..2970
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3030..3050
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3069..3359
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             3429..3467
                     /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             3486..3542
                     /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             3570..4271
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    4279..4867
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4332..4352)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4750..4761)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5015..5195
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5242..5260)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5328..5432
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5433..5501,5502..6293)
                     /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(5651..5670)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6464..7052
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7206..7223
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7463..7484
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7499..7504,7505..7522,7523..7529)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7537..7553
                     /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     7542..7564
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7561..7577
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7561..7577
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7627..7646
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 cttagtagtt ggaagggcta attcactccc aaagaagaca agatatcctt gatctgtgga
       61 tctaccacac acaaggctac ttccctgatt agcagaacta cacaccaggg ccaggggtca
      121 gatatccact gacctttgga tggtgctaca agctagtacc agttgagcca gataaggtag
      181 aagaggccaa taaaggagag aacaccagct tgttacaccc tgtgagcctg catgggatgg
      241 atgacccgga gagagaagtg ttagagtgga ggtttgacag ccgcctagca tttcatcacg
      301 tggcccgaga gctgcatccg gagtacttca agaactgctg atatcgagct tgctacaagg
      361 gactttccgc tggggacttt ccagggaggc gtggcctggg cgggactggg gagtggcgag
      421 ccctcagatc ctgcatataa gcagctgctt tttgcctgta ctgggtctct ctggttagac
      481 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
      541 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
      601 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg
      661 acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag
      721 cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg
      781 gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat
      841 cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata
      901 tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat
      961 cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag
     1021 aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga
     1081 taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca
     1141 ccgcacagca agcggccggc cgctgatctt cagacctgga ggaggagata tgagggacaa
     1201 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
     1261 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
     1321 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
     1381 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
     1441 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
     1501 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
     1561 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
     1621 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
     1681 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
     1741 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
     1801 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
     1861 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
     1921 gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg
     1981 agagagagac agagacagat ccattcgatt agtgaacgga tctcgacggt atcgccgaat
     2041 tcacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     2101 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     2161 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg
     2221 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
     2281 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     2341 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     2401 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     2461 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     2521 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     2581 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
     2641 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     2701 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
     2761 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctccctatc
     2821 agtgatagag atctccctat cagtgataga gatcgtcgac gagctcgttt agtgaaccgt
     2881 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga
     2941 tccagcctcc ggctagagcg gccgccatgg gatccggttc tgctagcggt tcaggttcta
     3001 ccggtggatc atctagaact agcggtagcg agaatttgta ttttcagggt ggtagcggat
     3061 ctaccggatc ggactcagaa gtcaatcaag aagctaagcc agaggtcaag ccagaagtca
     3121 agcctgagac tcacatcaat ttaaaggtgt ccgatggatc ttcagaaatc ttcttcaaga
     3181 tcaaaaagac cactccttta agaaggctga tggaagcgtt cgctaaaaga cagggtaagg
     3241 aaatggactc tttaaccttc ttgtacgacg gtattgagat tcaagctgat cagacccctg
     3301 aagatttgga catggaggat aacgatatta ttgaggctca cagagaacag attggtggcg
     3361 gatctggaag tggatctgga tcaggaagtg gttctggatc aggtagtggt tctggtagtg
     3421 gatcaccatc tcgcctggaa gaggaactgc gtcgtcgcct gaccgaacca accggaggaa
     3481 gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac
     3541 ctggaacggg cggagctggt tcaacgtcta tgagcgagct gattaaggag aacatgcaca
     3601 tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg
     3661 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc
     3721 tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca
     3781 accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg
     3841 agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc
     3901 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc
     3961 ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg
     4021 acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga
     4081 tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg
     4141 gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg
     4201 tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca
     4261 agcttaatta actcgagtaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     4321 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     4381 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     4441 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     4501 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     4561 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     4621 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgttggg gaaatcatcg
     4681 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     4741 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     4801 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     4861 tccccgcctg gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     4921 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatcacct
     4981 gcaggacagg cgcgccctgc tttttgcttg tactgggtct ctctggttag accagatctg
     5041 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     5101 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     5161 cagacccttt tagtcagtgt ggaaaatctc tagcacccgg gcgattaagg aaagggctag
     5221 atcattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     5281 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5341 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5401 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5461 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5521 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5581 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5641 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     5701 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5761 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5821 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5881 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5941 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6001 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6061 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6121 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6181 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6241 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6301 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6361 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6421 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6481 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6541 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6601 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6661 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6721 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6781 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6841 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6901 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6961 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7021 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7081 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     7141 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     7201 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     7261 ccgcgcgttg gccgattcat taatgcagca agctcatggc tgactaattt tttttattta
     7321 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     7381 tggaggccta ggcttttgca aaaagctccc cgtggcacga caggtttccc gactggaaag
     7441 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     7501 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     7561 caggaaacag ctatgacatg attacgaatt tcacaaataa agcatttttt tcactgcatt
     7621 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc aactggataa
     7681 ctcaagctaa ccaaaatcat cccaaacttc ccaccccata ccctattacc actgccaatt
     7741 acctgtggtt tcatttactc taaacctgtg attcctctga attattttca ttttaaagaa
     7801 attgtatttg ttaaatatgt actacaaa
//
