LOCUS       sequence-393118-        7243 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for constitutive expression of a 
            TagBFP-3xFLAG in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7243)
  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 7243)
  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: pTP341 pHAGE2 CMV TagBFP-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7243
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    680..805
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1302..1535
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1720..1764
                     /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    2057..2174
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2226..2529
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2530..2733
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2684..2704
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2730..2754
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2882..3580
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             3602..3667
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKEHDGDYKDHDIDYKDDDDK"
     misc_feature    3684..4272
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3737..3757)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4155..4166)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4420..4600
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4647..4665)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4733..4837
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4838..4906,4907..5698)
                     /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(5056..5075)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5869..6457
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6611..6628
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6868..6889
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6904..6909,6910..6927,6928..6934)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6942..6958
                     /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     6947..6969
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6966..6982
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6966..6982
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7032..7051
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(7243,1..633)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 ggaagggcta attcactccc aaagaagaca agatatcctt gatctgtgga tctaccacac
       61 acaaggctac ttccctgatt agcagaacta cacaccaggg ccaggggtca gatatccact
      121 gacctttgga tggtgctaca agctagtacc agttgagcca gataaggtag aagaggccaa
      181 taaaggagag aacaccagct tgttacaccc tgtgagcctg catgggatgg atgacccgga
      241 gagagaagtg ttagagtgga ggtttgacag ccgcctagca tttcatcacg tggcccgaga
      301 gctgcatccg gagtacttca agaactgctg atatcgagct tgctacaagg gactttccgc
      361 tggggacttt ccagggaggc gtggcctggg cgggactggg gagtggcgag ccctcagatc
      421 ctgcatataa gcagctgctt tttgcctgta ctgggtctct ctggttagac cagatctgag
      481 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
      541 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
      601 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
      661 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
      721 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
      781 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
      841 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
      901 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
      961 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
     1021 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
     1081 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     1141 agcggccggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     1201 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1261 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1321 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1381 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1441 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1501 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1561 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1621 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1681 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1741 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1801 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1861 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1921 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1981 agagacagat ccattcgatt agtgaacgga tctcgacggt atcgccgaat tcacaaatgg
     2041 cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa
     2101 gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa
     2161 aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg actagtggag
     2221 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     2281 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     2341 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     2401 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     2461 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     2521 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
     2581 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
     2641 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
     2701 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
     2761 agacgccatc cacgctgttt tgacctccat agaagacacc ggctagcggt tcgaccaagc
     2821 ttacggtacc atgggatcca ccggtggttc aggaagtact gcggccgctg gttcaacgtc
     2881 tatgagcgag ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga
     2941 caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac
     3001 catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac
     3061 tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt
     3121 caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg
     3181 cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa
     3241 gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg
     3301 ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat
     3361 ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc
     3421 caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga
     3481 aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag
     3541 atactgcgac ctccctagca aactggggca caagcttaat ggatcttcag gaggtagtat
     3601 ggactacaaa gagcatgacg gtgattataa agatcatgac atcgattaca aggatgacga
     3661 tgacaaggcc taatagctcg agtaatcaac ctctggatta caaaatttgt gaaagattga
     3721 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     3781 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     3841 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     3901 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     3961 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     4021 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg ttggggaaat
     4081 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     4141 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     4201 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     4261 ccgcctcccc gcctggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
     4321 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     4381 cacctgcagg acaggcgcgc cctgcttttt gcttgtactg ggtctctctg gttagaccag
     4441 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     4501 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     4561 tccctcagac ccttttagtc agtgtggaaa atctctagca cccgggcgat taaggaaagg
     4621 gctagatcat tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4681 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4741 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4801 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4861 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4921 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4981 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     5041 cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca
     5101 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     5161 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     5221 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     5281 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     5341 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     5401 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     5461 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5521 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5581 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5641 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5701 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5761 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5821 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5881 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5941 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     6001 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     6061 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     6121 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     6181 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     6241 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     6301 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     6361 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     6421 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     6481 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     6541 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     6601 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     6661 tctccccgcg cgttggccga ttcattaatg cagcaagctc atggctgact aatttttttt
     6721 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     6781 ttttttggag gcctaggctt ttgcaaaaag ctccccgtgg cacgacaggt ttcccgactg
     6841 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     6901 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     6961 tcacacagga aacagctatg acatgattac gaatttcaca aataaagcat ttttttcact
     7021 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcaactg
     7081 gataactcaa gctaaccaaa atcatcccaa acttcccacc ccatacccta ttaccactgc
     7141 caattacctg tggtttcatt tactctaaac ctgtgattcc tctgaattat tttcatttta
     7201 aagaaattgt atttgttaaa tatgtactac aaacttagta gtt
//
