LOCUS       sequence-393686-        7522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a N-terminally BFP-P2A-ALFA-TEV-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7522)
  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 7522)
  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: pTS100 pHAGE2 CMVtetO2 TagBFP-P2A-ALFA-22xGS-T...
FEATURES             Location/Qualifiers
     source          1..7522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2219..2598
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2599..2802
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2753..2773
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2804..2822
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2825..2843
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2853..2877
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2952..2961
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2964..3662
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             3684..3740
                     /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             3759..3797
                     /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             3873..3893
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     misc_feature    3964..4552
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4017..4037)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4435..4446)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4700..4880
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4927..4945)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5013..5117
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5118..5186,5187..5978)
                     /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(5336..5355)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6149..6737
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6891..6908
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7148..7169
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7184..7189,7190..7207,7208..7214)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7222..7238
                     /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     7227..7249
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7246..7262
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7246..7262
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7312..7331
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gacgcgttga
     2221 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     2281 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     2341 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     2401 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     2461 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     2521 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     2581 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     2641 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     2701 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     2761 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctccctat cagtgataga
     2821 gatctcccta tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg tcagatcgcc
     2881 tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc
     2941 cggctagagc ggccgccatg ggtatgagcg agctgattaa ggagaacatg cacatgaagc
     3001 tgtacatgga gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca
     3061 agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct
     3121 tcgccttcga catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca
     3181 cccagggcat ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag
     3241 tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
     3301 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga
     3361 tgcagaagaa aacactcggc tgggaggcct tcaccgagac gctgtacccc gctgacggcg
     3421 gcctggaagg cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa
     3481 acatcaagac cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtct
     3541 actatgtgga ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc
     3601 agcacgaggt ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctta
     3661 atactggtac cggaggaagc ggagctacta acttcagcct gctgaagcag gctggagacg
     3721 tggaggagaa ccctggacct ggagctgctg cagcgccatc tcgcctggaa gaggaactgc
     3781 gtcgtcgcct gaccgaacca ggatctggca gtggatcagg atctggtagt ggttctggta
     3841 gcggatctgg aagtggcagt ggatctggta gcgagaattt gtattttcag ggtggatctt
     3901 ctagtggatc cggttctgct agcggttcag gttctaccgg tggatcatct agataactcg
     3961 agtaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     4021 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     4081 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     4141 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     4201 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     4261 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     4321 ctgttgggca ctgacaattc cgtggtgttg ttggggaaat catcgtcctt tccttggctg
     4381 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     4441 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     4501 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcctggtacc
     4561 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     4621 gggactggaa gggctaattc actcccaacg aagacaagat cacctgcagg acaggcgcgc
     4681 cctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     4741 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     4801 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     4861 agtgtggaaa atctctagca cccgggcgat taaggaaagg gctagatcat tcttgaagac
     4921 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     4981 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5041 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5101 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5161 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5221 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5281 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5341 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     5401 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5461 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5521 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5581 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5641 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5701 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5761 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5821 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5881 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5941 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6001 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6061 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6121 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6181 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6241 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6301 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6361 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6421 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6481 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6541 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6601 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6661 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6721 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6781 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6841 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     6901 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     6961 ttcattaatg cagcaagctc atggctgact aatttttttt atttatgcag aggccgaggc
     7021 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     7081 ttgcaaaaag ctccccgtgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     7141 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     7201 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     7261 acatgattac gaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     7321 ccaaactcat caatgtatct tatcatgtct ggatcaactg gataactcaa gctaaccaaa
     7381 atcatcccaa acttcccacc ccatacccta ttaccactgc caattacctg tggtttcatt
     7441 tactctaaac ctgtgattcc tctgaattat tttcatttta aagaaattgt atttgttaaa
     7501 tatgtactac aaacttagta gt
//
