LOCUS       sequence-389917-        7528 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a C-terminally 3C-ALFA-P2A-BFP-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7528)
  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 7528)
  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: pTS111 pHAGE2 CMVtetO2 MCS-3C-22xGS-Alfa-P2A-T...
FEATURES             Location/Qualifiers
     source          1..7528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    676..801
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1298..1531
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1716..1760
                     /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    2053..2170
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2214..2593
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2594..2797
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2748..2768
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2799..2817
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2820..2838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2848..2872
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2947..2956
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3016..3039
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3115..3153
                     /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             3172..3228
                     /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             3256..3957
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    3965..4553
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4018..4038)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4436..4447)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4701..4881
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4928..4946)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5014..5118
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5119..5187,5188..5979)
                     /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(5337..5356)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6150..6738
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6892..6909
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7149..7170
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7185..7190,7191..7208,7209..7215)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7223..7239
                     /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     7228..7250
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7247..7263
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7247..7263
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7313..7332
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(7524..7528,1..629)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 gggctaattc actcccaaag aagacaagat atccttgatc tgtggatcta ccacacacaa
       61 ggctacttcc ctgattagca gaactacaca ccagggccag gggtcagata tccactgacc
      121 tttggatggt gctacaagct agtaccagtt gagccagata aggtagaaga ggccaataaa
      181 ggagagaaca ccagcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga
      241 gaagtgttag agtggaggtt tgacagccgc ctagcatttc atcacgtggc ccgagagctg
      301 catccggagt acttcaagaa ctgctgatat cgagcttgct acaagggact ttccgctggg
      361 gactttccag ggaggcgtgg cctgggcggg actggggagt ggcgagccct cagatcctgc
      421 atataagcag ctgctttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg
      481 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
      541 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
      601 cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa
      661 gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga
      721 ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag
      781 agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa
      841 attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc
      901 agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga
      961 caaatactgg gacagctaca accatccctt cagacaggat cagaagaact tagatcatta
     1021 tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag
     1081 gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg
     1141 gccggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
     1201 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
     1261 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
     1321 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
     1381 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
     1441 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
     1501 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
     1561 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
     1621 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
     1681 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
     1741 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
     1801 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
     1861 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
     1921 ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag
     1981 acagatccat tcgattagtg aacggatctc gacggtatcg ccgaattcac aaatggcagt
     2041 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     2101 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     2161 tcaaaatttt cgggtttatt acagggacag cagagatcca gtttggacgc gttgacattg
     2221 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     2281 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     2341 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     2401 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     2461 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     2521 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     2581 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     2641 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     2701 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     2761 taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg atagagatct
     2821 ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag
     2881 acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccggct
     2941 agagcggccg ccatgggatc cggttctgct agcggttcag gttctaccgg tggatcatct
     3001 agaactagcg gtagcttaga agttttgttt caaggtccag gatctggatc tggaagtgga
     3061 tctggatcag gaagtggttc tggatcaggt agtggttctg gtagtggatc accatctcgc
     3121 ctggaagagg aactgcgtcg tcgcctgacc gaaccaaccg gaggaagcgg agctactaac
     3181 ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctgg aacgggcgga
     3241 gctggttcaa cgtctatgag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg
     3301 gagggcaccg tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac
     3361 gagggcaccc agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc
     3421 gacatcctgg ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc
     3481 atccccgact tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca
     3541 tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc
     3601 atctacaacg tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag
     3661 aaaacactcg gctgggaggc cttcaccgag acgctgtacc ccgctgacgg cggcctggaa
     3721 ggcagaaacg acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacatcaag
     3781 accacatata gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg
     3841 gactacagac tggaaagaat caaggaggcc aacaacgaga cctacgtcga gcagcacgag
     3901 gtggcagtgg ccagatactg cgacctccct agcaaactgg ggcacaagct taattaactc
     3961 gagtaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     4021 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     4081 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     4141 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     4201 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     4261 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     4321 gctgttgggc actgacaatt ccgtggtgtt gttggggaaa tcatcgtcct ttccttggct
     4381 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     4441 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     4501 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcctggtac
     4561 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     4621 ggggactgga agggctaatt cactcccaac gaagacaaga tcacctgcag gacaggcgcg
     4681 ccctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     4741 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     4801 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     4861 cagtgtggaa aatctctagc acccgggcga ttaaggaaag ggctagatca ttcttgaaga
     4921 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     4981 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5041 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5101 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5161 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5221 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5281 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5341 tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac
     5401 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5461 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5521 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5581 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5641 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5701 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5761 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5821 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5881 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     5941 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6001 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6061 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6121 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6181 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6241 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6301 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6361 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6421 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6481 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6541 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6601 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6661 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6721 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6781 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     6841 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     6901 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     6961 attcattaat gcagcaagct catggctgac taattttttt tatttatgca gaggccgagg
     7021 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     7081 tttgcaaaaa gctccccgtg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     7141 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     7201 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     7261 gacatgatta cgaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     7321 tccaaactca tcaatgtatc ttatcatgtc tggatcaact ggataactca agctaaccaa
     7381 aatcatccca aacttcccac cccataccct attaccactg ccaattacct gtggtttcat
     7441 ttactctaaa cctgtgattc ctctgaatta ttttcatttt aaagaaattg tatttgttaa
     7501 atatgtacta caaacttagt agttggaa
//
