LOCUS       sequence-389910-        7822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a C-terminally 3C-SUMOEu-ALFA-P2A-BFP-tagged protein in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7822)
  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 7822)
  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: pTS113 pHAGE2 CMVtetO2
            MCS-3C-SUMOEu-22xGS-Alf...
FEATURES             Location/Qualifiers
     source          1..7822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    669..794
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1291..1524
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1709..1753
                     /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    2046..2163
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2207..2586
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2587..2790
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2741..2761
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2792..2810
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2813..2831
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2841..2865
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2940..2949
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3009..3032
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3402..3440
                     /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             3459..3515
                     /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             3543..4244
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    4252..4840
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4305..4325)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4723..4734)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4988..5168
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5215..5233)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5301..5405
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5406..5474,5475..6266)
                     /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(5624..5643)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6437..7025
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7179..7196
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7436..7457
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7472..7477,7478..7495,7496..7502)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7510..7526
                     /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     7515..7537
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7534..7550
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7534..7550
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7600..7619
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(7811..7822,1..622)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 ttcactccca aagaagacaa gatatccttg atctgtggat ctaccacaca caaggctact
       61 tccctgatta gcagaactac acaccagggc caggggtcag atatccactg acctttggat
      121 ggtgctacaa gctagtacca gttgagccag ataaggtaga agaggccaat aaaggagaga
      181 acaccagctt gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtgt
      241 tagagtggag gtttgacagc cgcctagcat ttcatcacgt ggcccgagag ctgcatccgg
      301 agtacttcaa gaactgctga tatcgagctt gctacaaggg actttccgct ggggactttc
      361 cagggaggcg tggcctgggc gggactgggg agtggcgagc cctcagatcc tgcatataag
      421 cagctgcttt ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
      481 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
      541 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
      601 tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac
      661 cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg
      721 ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg
      781 ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt
      841 taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc
      901 tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac
      961 tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata
     1021 cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt
     1081 tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc
     1141 gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg aattatataa
     1201 atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt
     1261 ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc
     1321 agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt
     1381 gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct
     1441 gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag
     1501 atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac
     1561 cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca
     1621 cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt
     1681 aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa
     1741 atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt
     1801 cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt
     1861 gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag
     1921 gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc
     1981 cattcgatta gtgaacggat ctcgacggta tcgccgaatt cacaaatggc agtattcatc
     2041 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     2101 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     2161 tttcgggttt attacaggga cagcagagat ccagtttgga cgcgttgaca ttgattattg
     2221 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     2281 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     2341 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     2401 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     2461 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     2521 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     2581 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     2641 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     2701 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     2761 gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc
     2821 agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat
     2881 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg gctagagcgg
     2941 ccgccatggg atccggttct gctagcggtt caggttctac cggtggatca tctagaacta
     3001 gcggtagctt agaagttttg tttcaaggtc caggtagcgg atcttctgct gcgggtggcg
     3061 aagaagataa gaaaccggca ggtggcgaag gtggcggtgc ccatatcaac ctgaaagtga
     3121 aaggtcaaga cggcaacgaa gtctttttcc gcatcaaacg ttctacccag ctgaaaaagc
     3181 tgatgaacgc atactgtgac cgtcagtctg tagacatgaa ggcaattgct ttcctcttta
     3241 agggtcgtcg cctacgtgcg gaaaggaccc cggatgaact ggaaatggaa gatggcgacg
     3301 aaatcgacgc aatgctgcat cagactggtg gcggatctgg aagtggatct ggatcaggaa
     3361 gtggttctgg atcaggtagt ggttctggta gtggatcacc atctcgcctg gaagaggaac
     3421 tgcgtcgtcg cctgaccgaa ccaaccggag gaagcggagc tactaacttc agcctgctga
     3481 agcaggctgg agacgtggag gagaaccctg gacctggaac gggcggagct ggttcaacgt
     3541 ctatgagcga gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtgg
     3601 acaaccatca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga
     3661 ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta
     3721 ctagcttcct ctacggcagc aagaccttca tcaaccacac ccagggcatc cccgacttct
     3781 tcaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg
     3841 gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca
     3901 agatcagagg ggtgaacttc acatccaacg gccctgtgat gcagaagaaa acactcggct
     3961 gggaggcctt caccgagacg ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca
     4021 tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa catcaagacc acatatagat
     4081 ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac tacagactgg
     4141 aaagaatcaa ggaggccaac aacgagacct acgtcgagca gcacgaggtg gcagtggcca
     4201 gatactgcga cctccctagc aaactggggc acaagcttaa ttaactcgag taatcaacct
     4261 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     4321 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     4381 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     4441 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     4501 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     4561 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     4621 gacaattccg tggtgttgtt ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     4681 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     4741 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     4801 cctcagacga gtcggatctc cctttgggcc gcctccccgc ctggtacctt taagaccaat
     4861 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
     4921 gctaattcac tcccaacgaa gacaagatca cctgcaggac aggcgcgccc tgctttttgc
     4981 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     5041 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     5101 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     5161 ctctagcacc cgggcgatta aggaaagggc tagatcattc ttgaagacga aagggcctcg
     5221 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     5281 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5341 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5401 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5461 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5521 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5581 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5641 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5701 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5761 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5821 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5881 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5941 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6001 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6061 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6121 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6181 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6241 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6301 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6361 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6421 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6481 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6541 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6601 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6661 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6721 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6781 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6841 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     6901 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     6961 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7021 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7081 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     7141 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     7201 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     7261 gcaagctcat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     7321 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     7381 ccccgtggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     7441 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     7501 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac atgattacga
     7561 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     7621 atgtatctta tcatgtctgg atcaactgga taactcaagc taaccaaaat catcccaaac
     7681 ttcccacccc ataccctatt accactgcca attacctgtg gtttcattta ctctaaacct
     7741 gtgattcctc tgaattattt tcattttaaa gaaattgtat ttgttaaata tgtactacaa
     7801 acttagtagt tggaagggct aa
//
