LOCUS       sequence-389904-        7777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a N-terminally BFP-P2A-ALFA-SUMOEu-tagged protein in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7777)
  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 7777)
  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: pTS101 pHAGE2 CMVtetO2
            TagBFP-P2A-ALFA-22xGS-S...
FEATURES             Location/Qualifiers
     source          1..7777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             14..647
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    694..819
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1316..1549
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1734..1778
                     /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    2071..2188
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2232..2611
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2612..2815
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2766..2786
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2817..2835
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2838..2856
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2866..2890
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2965..2974
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2977..3675
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             3697..3753
                     /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             3772..3810
                     /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"
     misc_feature    4232..4820
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4285..4305)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4703..4714)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4968..5148
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5195..5213)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5281..5385
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5386..5454,5455..6246)
                     /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(5604..5623)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6417..7005
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7159..7176
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7416..7437
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7452..7457,7458..7475,7476..7482)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7490..7506
                     /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     7495..7517
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7514..7530
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7514..7530
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7580..7599
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 caaacttagt agttggaagg gctaattcac tcccaaagaa gacaagatat ccttgatctg
       61 tggatctacc acacacaagg ctacttccct gattagcaga actacacacc agggccaggg
      121 gtcagatatc cactgacctt tggatggtgc tacaagctag taccagttga gccagataag
      181 gtagaagagg ccaataaagg agagaacacc agcttgttac accctgtgag cctgcatggg
      241 atggatgacc cggagagaga agtgttagag tggaggtttg acagccgcct agcatttcat
      301 cacgtggccc gagagctgca tccggagtac ttcaagaact gctgatatcg agcttgctac
      361 aagggacttt ccgctgggga ctttccaggg aggcgtggcc tgggcgggac tggggagtgg
      421 cgagccctca gatcctgcat ataagcagct gctttttgcc tgtactgggt ctctctggtt
      481 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
      541 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
      601 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
      661 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
      721 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
      781 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
      841 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
      901 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
      961 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
     1021 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
     1081 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
     1141 accaccgcac agcaagcggc cggccgctga tcttcagacc tggaggagga gatatgaggg
     1201 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1261 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1321 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     1381 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     1441 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     1501 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     1561 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     1621 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     1681 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     1741 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     1801 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     1861 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     1921 ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag
     1981 gtggagagag agacagagac agatccattc gattagtgaa cggatctcga cggtatcgcc
     2041 gaattcacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     2101 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     2161 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt
     2221 ttggacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     2281 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     2341 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     2401 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     2461 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     2521 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     2581 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     2641 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     2701 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     2761 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctccc
     2821 tatcagtgat agagatctcc ctatcagtga tagagatcgt cgacgagctc gtttagtgaa
     2881 ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga
     2941 ccgatccagc ctccggctag agcggccgcc atgggtatga gcgagctgat taaggagaac
     3001 atgcacatga agctgtacat ggagggcacc gtggacaacc atcacttcaa gtgcacatcc
     3061 gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggt ggtcgagggc
     3121 ggccctctcc ccttcgcctt cgacatcctg gctactagct tcctctacgg cagcaagacc
     3181 ttcatcaacc acacccaggg catccccgac ttcttcaagc agtccttccc tgagggcttc
     3241 acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac ccaggacacc
     3301 agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa cttcacatcc
     3361 aacggccctg tgatgcagaa gaaaacactc ggctgggagg ccttcaccga gacgctgtac
     3421 cccgctgacg gcggcctgga aggcagaaac gacatggccc tgaagctcgt gggcgggagc
     3481 catctgatcg caaacatcaa gaccacatat agatccaaga aacccgctaa gaacctcaag
     3541 atgcctggcg tctactatgt ggactacaga ctggaaagaa tcaaggaggc caacaacgag
     3601 acctacgtcg agcagcacga ggtggcagtg gccagatact gcgacctccc tagcaaactg
     3661 gggcacaagc ttaatactgg taccggagga agcggagcta ctaacttcag cctgctgaag
     3721 caggctggag acgtggagga gaaccctgga cctggagctg ctgcagcgcc atctcgcctg
     3781 gaagaggaac tgcgtcgtcg cctgaccgaa ccaggatctg gcagtggatc aggatctggt
     3841 agtggttctg gtagcggatc tggaagtggc agtggatctt ctgctgcggg tggcgaagaa
     3901 gataagaaac cggcaggtgg cgaaggtggc ggtgcccata tcaacctgaa agtgaaaggt
     3961 caagacggca acgaagtctt tttccgcatc aaacgttcta cccagctgaa aaagctgatg
     4021 aacgcatact gtgaccgtca gtctgtagac atgaaggcaa ttgctttcct ctttaagggt
     4081 cgtcgcctac gtgcggaaag gaccccggat gaactggaaa tggaagatgg cgacgaaatc
     4141 gacgcaatgc tgcatcagac tggtggctct agtggatccg gttctgctag cggttcaggt
     4201 tctaccggtg gatcatctag ataactcgag taatcaacct ctggattaca aaatttgtga
     4261 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     4321 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     4381 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     4441 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     4501 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     4561 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtt
     4621 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     4681 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     4741 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     4801 cctttgggcc gcctccccgc ctggtacctt taagaccaat gacttacaag gcagctgtag
     4861 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa
     4921 gacaagatca cctgcaggac aggcgcgccc tgctttttgc ttgtactggg tctctctggt
     4981 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     5041 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     5101 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcacc cgggcgatta
     5161 aggaaagggc tagatcattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     5221 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     5281 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5341 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5401 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5461 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5521 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5581 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     5641 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5701 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5761 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5821 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5881 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5941 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6001 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6061 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6121 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6181 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6241 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6301 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6361 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6421 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6481 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6541 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6601 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6661 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6721 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6781 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6841 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6901 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6961 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     7021 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     7081 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     7141 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     7201 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gcaagctcat ggctgactaa
     7261 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     7321 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct ccccgtggca cgacaggttt
     7381 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     7441 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     7501 taacaatttc acacaggaaa cagctatgac atgattacga atttcacaaa taaagcattt
     7561 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     7621 atcaactgga taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt
     7681 accactgcca attacctgtg gtttcattta ctctaaacct gtgattcctc tgaattattt
     7741 tcattttaaa gaaattgtat ttgttaaata tgtacta
//
