LOCUS       sequence-389906-        7525 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a C-terminally TEV-ALFA-P2A-BFP-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: pTS112 pHAGE2 CMVtetO2 MCS-TEV-22xGS-Alfa-P2A-...
FEATURES             Location/Qualifiers
     source          1..7525
                     /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             3021..3041
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3117..3155
                     /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             3174..3230
                     /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             3258..3959
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    3967..4555
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4020..4040)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4438..4449)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4703..4883
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4930..4948)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5016..5120
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5121..5189,5190..5981)
                     /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(5339..5358)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6152..6740
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6894..6911
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7151..7172
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7187..7192,7193..7210,7211..7217)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7225..7241
                     /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     7230..7252
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7249..7265
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7249..7265
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7315..7334
                     /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 ggatccggtt ctgctagcgg ttcaggttct accggtggat
     3001 catctagaac tagcggtagc gagaatttgt attttcaggg tggatctgga tctggaagtg
     3061 gatctggatc aggaagtggt tctggatcag gtagtggttc tggtagtgga tcaccatctc
     3121 gcctggaaga ggaactgcgt cgtcgcctga ccgaaccaac cggaggaagc ggagctacta
     3181 acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct ggaacgggcg
     3241 gagctggttc aacgtctatg agcgagctga ttaaggagaa catgcacatg aagctgtaca
     3301 tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct
     3361 acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct
     3421 tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg
     3481 gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca
     3541 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
     3601 tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga
     3661 agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg
     3721 aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacatca
     3781 agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg
     3841 tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg
     3901 aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattaac
     3961 tcgagtaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     4021 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     4081 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     4141 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     4201 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     4261 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     4321 cggctgttgg gcactgacaa ttccgtggtg ttgttgggga aatcatcgtc ctttccttgg
     4381 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     4441 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     4501 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctggt
     4561 acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa
     4621 ggggggactg gaagggctaa ttcactccca acgaagacaa gatcacctgc aggacaggcg
     4681 cgccctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct
     4741 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     4801 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     4861 gtcagtgtgg aaaatctcta gcacccgggc gattaaggaa agggctagat cattcttgaa
     4921 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     4981 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5041 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5101 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5161 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5221 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5281 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5341 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     5401 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5461 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5521 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5581 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5641 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5701 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5761 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5821 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5881 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5941 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6001 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6061 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6121 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6181 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6241 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6301 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6361 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6421 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6481 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6541 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6601 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6661 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6721 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6781 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6841 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     6901 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     6961 cgattcatta atgcagcaag ctcatggctg actaattttt tttatttatg cagaggccga
     7021 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     7081 cttttgcaaa aagctccccg tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     7141 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     7201 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     7261 atgacatgat tacgaatttc acaaataaag catttttttc actgcattct agttgtggtt
     7321 tgtccaaact catcaatgta tcttatcatg tctggatcaa ctggataact caagctaacc
     7381 aaaatcatcc caaacttccc accccatacc ctattaccac tgccaattac ctgtggtttc
     7441 atttactcta aacctgtgat tcctctgaat tattttcatt ttaaagaaat tgtatttgtt
     7501 aaatatgtac tacaaactta gtagt
//
