LOCUS       sequence-389911-        7819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a C-terminally TEV-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 7819)
  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 7819)
  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: pTS114 pHAGE2 CMVtetO2
            MCS-TEV-SUMOEu-22xGS-Al...
FEATURES             Location/Qualifiers
     source          1..7819
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    649..774
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1271..1504
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1689..1733
                     /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    2026..2143
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2187..2566
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2567..2770
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2721..2741
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2772..2790
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2793..2811
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2821..2845
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2920..2929
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2989..3009
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3379..3417
                     /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             3436..3492
                     /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             3520..4221
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    4229..4817
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4282..4302)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4700..4711)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4965..5145
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5192..5210)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5278..5382
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5383..5451,5452..6243)
                     /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(5601..5620)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6414..7002
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7156..7173
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7413..7434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7449..7454,7455..7472,7473..7479)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7487..7503
                     /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     7492..7514
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7511..7527
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7511..7527
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7577..7596
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(7788..7819,1..602)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 gatatccttg atctgtggat ctaccacaca caaggctact tccctgatta gcagaactac
       61 acaccagggc caggggtcag atatccactg acctttggat ggtgctacaa gctagtacca
      121 gttgagccag ataaggtaga agaggccaat aaaggagaga acaccagctt gttacaccct
      181 gtgagcctgc atgggatgga tgacccggag agagaagtgt tagagtggag gtttgacagc
      241 cgcctagcat ttcatcacgt ggcccgagag ctgcatccgg agtacttcaa gaactgctga
      301 tatcgagctt gctacaaggg actttccgct ggggactttc cagggaggcg tggcctgggc
      361 gggactgggg agtggcgagc cctcagatcc tgcatataag cagctgcttt ttgcctgtac
      421 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
      481 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
      541 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
      601 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
      661 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
      721 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
      781 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
      841 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
      901 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
      961 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     1021 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     1081 caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc gctgatcttc agacctggag
     1141 gaggagatat gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg
     1201 aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag
     1261 cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg
     1321 cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc
     1381 agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg
     1441 gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc
     1501 tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg
     1561 ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg
     1621 acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc
     1681 agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt
     1741 ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct
     1801 tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat
     1861 attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag
     1921 gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat
     1981 ctcgacggta tcgccgaatt cacaaatggc agtattcatc cacaatttta aaagaaaagg
     2041 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2101 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2161 cagcagagat ccagtttgga cgcgttgaca ttgattattg actagttatt aatagtaatc
     2221 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     2281 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     2341 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     2401 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     2461 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     2521 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     2581 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     2641 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     2701 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     2761 aagcagagct ctccctatca gtgatagaga tctccctatc agtgatagag atcgtcgacg
     2821 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca
     2881 tagaagacac cgggaccgat ccagcctccg gctagagcgg ccgccatggg atccggttct
     2941 gctagcggtt caggttctac cggtggatca tctagaacta gcggtagcga gaatttgtat
     3001 tttcagggtg gtagcggatc ttctgctgcg ggtggcgaag aagataagaa accggcaggt
     3061 ggcgaaggtg gcggtgccca tatcaacctg aaagtgaaag gtcaagacgg caacgaagtc
     3121 tttttccgca tcaaacgttc tacccagctg aaaaagctga tgaacgcata ctgtgaccgt
     3181 cagtctgtag acatgaaggc aattgctttc ctctttaagg gtcgtcgcct acgtgcggaa
     3241 aggaccccgg atgaactgga aatggaagat ggcgacgaaa tcgacgcaat gctgcatcag
     3301 actggtggcg gatctggaag tggatctgga tcaggaagtg gttctggatc aggtagtggt
     3361 tctggtagtg gatcaccatc tcgcctggaa gaggaactgc gtcgtcgcct gaccgaacca
     3421 accggaggaa gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag
     3481 aaccctggac ctggaacggg cggagctggt tcaacgtcta tgagcgagct gattaaggag
     3541 aacatgcaca tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca
     3601 tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag
     3661 ggcggccctc tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag
     3721 accttcatca accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc
     3781 ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac
     3841 accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca
     3901 tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg
     3961 taccccgctg acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg
     4021 agccatctga tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc
     4081 aagatgcctg gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac
     4141 gagacctacg tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa
     4201 ctggggcaca agcttaatta actcgagtaa tcaacctctg gattacaaaa tttgtgaaag
     4261 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     4321 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     4381 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     4441 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     4501 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     4561 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgttggg
     4621 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     4681 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     4741 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     4801 ttgggccgcc tccccgcctg gtacctttaa gaccaatgac ttacaaggca gctgtagatc
     4861 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     4921 aagatcacct gcaggacagg cgcgccctgc tttttgcttg tactgggtct ctctggttag
     4981 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     5041 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     5101 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcacccgg gcgattaagg
     5161 aaagggctag atcattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5221 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5281 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5341 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5401 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5461 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5521 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5581 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     5641 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5701 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5761 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5821 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     5881 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     5941 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6001 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6061 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6121 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6181 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6241 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6301 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6361 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6421 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6481 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6541 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6601 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6661 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6721 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6781 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6841 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6901 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6961 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7021 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     7081 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     7141 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     7201 ccgcctctcc ccgcgcgttg gccgattcat taatgcagca agctcatggc tgactaattt
     7261 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     7321 gaggcttttt tggaggccta ggcttttgca aaaagctccc cgtggcacga caggtttccc
     7381 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     7441 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     7501 caatttcaca caggaaacag ctatgacatg attacgaatt tcacaaataa agcatttttt
     7561 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     7621 aactggataa ctcaagctaa ccaaaatcat cccaaacttc ccaccccata ccctattacc
     7681 actgccaatt acctgtggtt tcatttactc taaacctgtg attcctctga attattttca
     7741 ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagttgg aagggctaat
     7801 tcactcccaa agaagacaa
//
