LOCUS       sequence-389902-        7786 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a N-terminally BFP-P2A-ALFA-SUMOstar-tagged protein in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7786)
  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 7786)
  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: pTS102 pHAGE2 CMVtetO2
            TagBFP-P2A-ALFA-22xGS-S...
FEATURES             Location/Qualifiers
     source          1..7786
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             3..636
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    683..808
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1305..1538
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1723..1767
                     /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    2060..2177
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2221..2600
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2601..2804
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2755..2775
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2806..2824
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2827..2845
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2855..2879
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2954..2963
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2966..3664
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             3686..3742
                     /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             3761..3799
                     /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             3875..4165
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGG"
     misc_feature    4230..4818
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4283..4303)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4701..4712)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4966..5146
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5193..5211)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5279..5383
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5384..5452,5453..6244)
                     /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(5602..5621)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6415..7003
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7157..7174
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7414..7435
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7450..7455,7456..7473,7474..7480)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7488..7504
                     /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     7493..7515
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7512..7528
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7512..7528
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7578..7597
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 gttggaaggg ctaattcact cccaaagaag acaagatatc cttgatctgt ggatctacca
       61 cacacaaggc tacttccctg attagcagaa ctacacacca gggccagggg tcagatatcc
      121 actgaccttt ggatggtgct acaagctagt accagttgag ccagataagg tagaagaggc
      181 caataaagga gagaacacca gcttgttaca ccctgtgagc ctgcatggga tggatgaccc
      241 ggagagagaa gtgttagagt ggaggtttga cagccgccta gcatttcatc acgtggcccg
      301 agagctgcat ccggagtact tcaagaactg ctgatatcga gcttgctaca agggactttc
      361 cgctggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc gagccctcag
      421 atcctgcata taagcagctg ctttttgcct gtactgggtc tctctggtta gaccagatct
      481 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
      541 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
      601 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
      661 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
      721 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
      781 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
      841 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
      901 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
      961 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     1021 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     1081 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     1141 gcaagcggcc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
     1201 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
     1261 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
     1321 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
     1381 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
     1441 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
     1501 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
     1561 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
     1621 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
     1681 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
     1741 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
     1801 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
     1861 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
     1921 ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga
     1981 gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgccg aattcacaaa
     2041 tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg
     2101 aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta
     2161 caaaaattca aaattttcgg gtttattaca gggacagcag agatccagtt tggacgcgtt
     2221 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     2281 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     2341 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     2401 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     2461 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     2521 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     2581 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     2641 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     2701 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     2761 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
     2821 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
     2881 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
     2941 tccggctaga gcggccgcca tgggtatgag cgagctgatt aaggagaaca tgcacatgaa
     3001 gctgtacatg gagggcaccg tggacaacca tcacttcaag tgcacatccg agggcgaagg
     3061 caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg gccctctccc
     3121 cttcgccttc gacatcctgg ctactagctt cctctacggc agcaagacct tcatcaacca
     3181 cacccagggc atccccgact tcttcaagca gtccttccct gagggcttca catgggagag
     3241 agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga
     3301 cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcacatcca acggccctgt
     3361 gatgcagaag aaaacactcg gctgggaggc cttcaccgag acgctgtacc ccgctgacgg
     3421 cggcctggaa ggcagaaacg acatggccct gaagctcgtg ggcgggagcc atctgatcgc
     3481 aaacatcaag accacatata gatccaagaa acccgctaag aacctcaaga tgcctggcgt
     3541 ctactatgtg gactacagac tggaaagaat caaggaggcc aacaacgaga cctacgtcga
     3601 gcagcacgag gtggcagtgg ccagatactg cgacctccct agcaaactgg ggcacaagct
     3661 taatactggt accggaggaa gcggagctac taacttcagc ctgctgaagc aggctggaga
     3721 cgtggaggag aaccctggac ctggagctgc tgcagcgcca tctcgcctgg aagaggaact
     3781 gcgtcgtcgc ctgaccgaac caggatctgg cagtggatca ggatctggta gtggttctgg
     3841 tagcggatct ggaagtggca gtggatctac cggatcggac tcagaagtca atcaagaagc
     3901 taagccagag gtcaagccag aagtcaagcc tgagactcac atcaatttaa aggtgtccga
     3961 tggatcttca gaaatcttct tcaagatcaa aaagaccact cctttaagaa ggctgatgga
     4021 agcgttcgct aaaagacagg gtaaggaaat ggactcttta accttcttgt acgacggtat
     4081 tgagattcaa gctgatcaga cccctgaaga tttggacatg gaggataacg atattattga
     4141 ggctcacaga gaacagattg gtggctctag tggatccggt tctgctagcg gttcaggttc
     4201 taccggtgga tcatctagat aactcgagta atcaacctct ggattacaaa atttgtgaaa
     4261 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     4321 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     4381 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     4441 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     4501 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     4561 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgttgg
     4621 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     4681 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     4741 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     4801 tttgggccgc ctccccgcct ggtaccttta agaccaatga cttacaaggc agctgtagat
     4861 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     4921 caagatcacc tgcaggacag gcgcgccctg ctttttgctt gtactgggtc tctctggtta
     4981 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     5041 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     5101 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcacccg ggcgattaag
     5161 gaaagggcta gatcattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt
     5221 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5281 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5341 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5401 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5461 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5521 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5581 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca
     5641 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5701 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5761 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5821 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5881 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5941 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6001 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6061 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6121 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6181 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6241 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6301 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6361 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6421 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6481 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6541 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6601 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6661 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6721 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6781 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6841 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6901 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6961 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7021 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7081 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7141 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     7201 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc aagctcatgg ctgactaatt
     7261 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     7321 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc ccgtggcacg acaggtttcc
     7381 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     7441 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     7501 acaatttcac acaggaaaca gctatgacat gattacgaat ttcacaaata aagcattttt
     7561 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     7621 caactggata actcaagcta accaaaatca tcccaaactt cccaccccat accctattac
     7681 cactgccaat tacctgtggt ttcatttact ctaaacctgt gattcctctg aattattttc
     7741 attttaaaga aattgtattt gttaaatatg tactacaaac ttagta
//
