LOCUS       sequence-389897-        7483 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a N-terminally BFP-P2A-ALFA-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7483)
  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 7483)
  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: pTS98 pHAGE2 CMVtetO2 TagBFP-P2A-ALFA-22xGS-MCS
FEATURES             Location/Qualifiers
     source          1..7483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             7..640
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    687..812
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1309..1542
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1727..1771
                     /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    2064..2181
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2225..2604
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2605..2808
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2759..2779
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2810..2828
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2831..2849
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2859..2883
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2958..2967
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2970..3668
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             3690..3746
                     /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             3765..3803
                     /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"
     misc_feature    3931..4519
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3984..4004)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4402..4413)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4667..4847
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4894..4912)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4980..5084
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5085..5153,5154..5945)
                     /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(5303..5322)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6116..6704
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6858..6875
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7115..7136
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7151..7156,7157..7174,7175..7181)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7189..7205
                     /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     7194..7216
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7213..7229
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7213..7229
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7279..7298
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 agtagttgga agggctaatt cactcccaaa gaagacaaga tatccttgat ctgtggatct
       61 accacacaca aggctacttc cctgattagc agaactacac accagggcca ggggtcagat
      121 atccactgac ctttggatgg tgctacaagc tagtaccagt tgagccagat aaggtagaag
      181 aggccaataa aggagagaac accagcttgt tacaccctgt gagcctgcat gggatggatg
      241 acccggagag agaagtgtta gagtggaggt ttgacagccg cctagcattt catcacgtgg
      301 cccgagagct gcatccggag tacttcaaga actgctgata tcgagcttgc tacaagggac
      361 tttccgctgg ggactttcca gggaggcgtg gcctgggcgg gactggggag tggcgagccc
      421 tcagatcctg catataagca gctgcttttt gcctgtactg ggtctctctg gttagaccag
      481 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
      541 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
      601 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
      661 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
      721 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
      781 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
      841 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
      901 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
      961 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
     1021 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
     1081 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
     1141 cacagcaagc ggccggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     1201 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     1261 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     1321 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     1381 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     1441 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     1501 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     1561 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     1621 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     1681 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     1741 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     1801 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     1861 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     1921 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
     1981 gagagacaga gacagatcca ttcgattagt gaacggatct cgacggtatc gccgaattca
     2041 caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca
     2101 ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa
     2161 attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc agtttggacg
     2221 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     2281 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     2341 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     2401 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     2461 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     2521 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     2581 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     2641 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     2701 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     2761 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt
     2821 gatagagatc tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag
     2881 atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc
     2941 agcctccggc tagagcggcc gccatgggta tgagcgagct gattaaggag aacatgcaca
     3001 tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg
     3061 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc
     3121 tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca
     3181 accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg
     3241 agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc
     3301 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc
     3361 ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg
     3421 acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga
     3481 tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg
     3541 gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg
     3601 tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca
     3661 agcttaatac tggtaccgga ggaagcggag ctactaactt cagcctgctg aagcaggctg
     3721 gagacgtgga ggagaaccct ggacctggag ctgctgcagc gccatctcgc ctggaagagg
     3781 aactgcgtcg tcgcctgacc gaaccaggat ctggcagtgg atcaggatct ggtagtggtt
     3841 ctggtagcgg atctggaagt ggcagttcta gtggatccgg ttctgctagc ggttcaggtt
     3901 ctaccggtgg atcatctaga taactcgagt aatcaacctc tggattacaa aatttgtgaa
     3961 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     4021 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     4081 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     4141 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     4201 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     4261 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgttg
     4321 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     4381 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     4441 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     4501 ctttgggccg cctccccgcc tggtaccttt aagaccaatg acttacaagg cagctgtaga
     4561 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     4621 acaagatcac ctgcaggaca ggcgcgccct gctttttgct tgtactgggt ctctctggtt
     4681 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     4741 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     4801 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaccc gggcgattaa
     4861 ggaaagggct agatcattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     4921 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     4981 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5041 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5101 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5161 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5221 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5281 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     5341 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5401 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5461 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5521 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5581 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5641 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5701 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5761 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5821 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5881 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5941 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6001 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6061 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6121 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6181 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6241 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6301 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6361 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6421 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6481 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6541 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6601 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6661 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6721 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6781 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     6841 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     6901 aaccgcctct ccccgcgcgt tggccgattc attaatgcag caagctcatg gctgactaat
     6961 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     7021 aggaggcttt tttggaggcc taggcttttg caaaaagctc cccgtggcac gacaggtttc
     7081 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     7141 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     7201 aacaatttca cacaggaaac agctatgaca tgattacgaa tttcacaaat aaagcatttt
     7261 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     7321 tcaactggat aactcaagct aaccaaaatc atcccaaact tcccacccca taccctatta
     7381 ccactgccaa ttacctgtgg tttcatttac tctaaacctg tgattcctct gaattatttt
     7441 cattttaaag aaattgtatt tgttaaatat gtactacaaa ctt
//
