LOCUS       sequence-389908-        7525 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a N-terminally BFP-P2A-ALFA-3C-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7525)
  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 7525)
  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: pTS99 pHAGE2 CMVtetO2 TagBFP-P2A-ALFA-22xGS-3C...
FEATURES             Location/Qualifiers
     source          1..7525
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             9..642
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    689..814
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1311..1544
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1729..1773
                     /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    2066..2183
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2227..2606
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2607..2810
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2761..2781
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2812..2830
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2833..2851
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2861..2885
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2960..2969
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2972..3670
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             3692..3748
                     /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             3767..3805
                     /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             3881..3904
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     misc_feature    3975..4563
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4028..4048)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4446..4457)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4711..4891
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4938..4956)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5024..5128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5129..5197,5198..5989)
                     /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(5347..5366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6160..6748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6902..6919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7159..7180
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7195..7200,7201..7218,7219..7225)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7233..7249
                     /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     7238..7260
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7257..7273
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7257..7273
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7323..7342
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 ttagtagttg gaagggctaa ttcactccca aagaagacaa gatatccttg atctgtggat
       61 ctaccacaca caaggctact tccctgatta gcagaactac acaccagggc caggggtcag
      121 atatccactg acctttggat ggtgctacaa gctagtacca gttgagccag ataaggtaga
      181 agaggccaat aaaggagaga acaccagctt gttacaccct gtgagcctgc atgggatgga
      241 tgacccggag agagaagtgt tagagtggag gtttgacagc cgcctagcat ttcatcacgt
      301 ggcccgagag ctgcatccgg agtacttcaa gaactgctga tatcgagctt gctacaaggg
      361 actttccgct ggggactttc cagggaggcg tggcctgggc gggactgggg agtggcgagc
      421 cctcagatcc tgcatataag cagctgcttt ttgcctgtac tgggtctctc tggttagacc
      481 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      541 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
      601 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
      661 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
      721 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
      781 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
      841 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
      901 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
      961 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     1021 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     1081 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     1141 cgcacagcaa gcggccggcc gctgatcttc agacctggag gaggagatat gagggacaat
     1201 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     1261 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     1321 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     1381 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     1441 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     1501 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     1561 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     1621 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     1681 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     1741 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     1801 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     1861 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     1921 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     1981 gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta tcgccgaatt
     2041 cacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg
     2101 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac
     2161 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccagtttgga
     2221 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     2281 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     2341 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     2401 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     2461 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     2521 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     2581 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     2641 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     2701 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     2761 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca
     2821 gtgatagaga tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc
     2881 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
     2941 ccagcctccg gctagagcgg ccgccatggg tatgagcgag ctgattaagg agaacatgca
     3001 catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca catccgaggg
     3061 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc
     3121 tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca agaccttcat
     3181 caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg gcttcacatg
     3241 ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct
     3301 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca catccaacgg
     3361 ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc tgtaccccgc
     3421 tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg ggagccatct
     3481 gatcgcaaac atcaagacca catatagatc caagaaaccc gctaagaacc tcaagatgcc
     3541 tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca acgagaccta
     3601 cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca aactggggca
     3661 caagcttaat actggtaccg gaggaagcgg agctactaac ttcagcctgc tgaagcaggc
     3721 tggagacgtg gaggagaacc ctggacctgg agctgctgca gcgccatctc gcctggaaga
     3781 ggaactgcgt cgtcgcctga ccgaaccagg atctggcagt ggatcaggat ctggtagtgg
     3841 ttctggtagc ggatctggaa gtggcagtgg atctggtagc ttagaagttt tgtttcaagg
     3901 tccaggatct tctagtggat ccggttctgc tagcggttca ggttctaccg gtggatcatc
     3961 tagataactc gagtaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     4021 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     4081 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     4141 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     4201 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     4261 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     4321 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gttggggaaa tcatcgtcct
     4381 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     4441 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     4501 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     4561 cgcctggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     4621 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tcacctgcag
     4681 gacaggcgcg ccctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     4741 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     4801 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     4861 cccttttagt cagtgtggaa aatctctagc acccgggcga ttaaggaaag ggctagatca
     4921 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     4981 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5041 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5101 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5161 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5221 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5281 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5341 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
     5401 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5461 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5521 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5581 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     5641 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     5701 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     5761 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     5821 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     5881 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     5941 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6001 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6061 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6121 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6181 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6241 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6301 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6361 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6421 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6481 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6541 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6601 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6661 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6721 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6781 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6841 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     6901 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     6961 gcgttggccg attcattaat gcagcaagct catggctgac taattttttt tatttatgca
     7021 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     7081 ggcctaggct tttgcaaaaa gctccccgtg gcacgacagg tttcccgact ggaaagcggg
     7141 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     7201 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     7261 aaacagctat gacatgatta cgaatttcac aaataaagca tttttttcac tgcattctag
     7321 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcaact ggataactca
     7381 agctaaccaa aatcatccca aacttcccac cccataccct attaccactg ccaattacct
     7441 gtggtttcat ttactctaaa cctgtgattc ctctgaatta ttttcatttt aaagaaattg
     7501 tatttgttaa atatgtacta caaac
//
