LOCUS       sequence-389915-        7486 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a C-terminally ALFA-P2A-BFP-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7486)
  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 7486)
  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: pTS110 pHAGE2 CMVtetO2 MCS-22xGS-Alfa-P2A-TagBFP
FEATURES             Location/Qualifiers
     source          1..7486
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             27..660
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    707..832
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1329..1562
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1747..1791
                     /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    2084..2201
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2245..2624
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2625..2828
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2779..2799
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2830..2848
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2851..2869
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2879..2903
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2978..2987
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3104..3142
                     /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             3161..3217
                     /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             3245..3946
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    3954..4542
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4007..4027)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4425..4436)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4690..4870
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4917..4935)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5003..5107
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5108..5176,5177..5968)
                     /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(5326..5345)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6139..6727
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6881..6898
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7138..7159
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7174..7179,7180..7197,7198..7204)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7212..7228
                     /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     7217..7239
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7236..7252
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7236..7252
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7302..7321
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 taaatatgta ctacaaactt agtagttgga agggctaatt cactcccaaa gaagacaaga
       61 tatccttgat ctgtggatct accacacaca aggctacttc cctgattagc agaactacac
      121 accagggcca ggggtcagat atccactgac ctttggatgg tgctacaagc tagtaccagt
      181 tgagccagat aaggtagaag aggccaataa aggagagaac accagcttgt tacaccctgt
      241 gagcctgcat gggatggatg acccggagag agaagtgtta gagtggaggt ttgacagccg
      301 cctagcattt catcacgtgg cccgagagct gcatccggag tacttcaaga actgctgata
      361 tcgagcttgc tacaagggac tttccgctgg ggactttcca gggaggcgtg gcctgggcgg
      421 gactggggag tggcgagccc tcagatcctg catataagca gctgcttttt gcctgtactg
      481 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
      541 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
      601 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
      661 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
      721 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
      781 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
      841 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
      901 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
      961 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
     1021 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
     1081 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
     1141 aaacaaaagt aagaccaccg cacagcaagc ggccggccgc tgatcttcag acctggagga
     1201 ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa
     1261 ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca
     1321 gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca
     1381 gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag
     1441 aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc
     1501 atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc
     1561 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct
     1621 agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac
     1681 agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag
     1741 caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg
     1801 tttaacataa caaattggct gtggtatata aaattattca taatgatagt aggaggcttg
     1861 gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat
     1921 tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga
     1981 atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatct
     2041 cgacggtatc gccgaattca caaatggcag tattcatcca caattttaaa agaaaagggg
     2101 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2161 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2221 gcagagatcc agtttggacg cgttgacatt gattattgac tagttattaa tagtaatcaa
     2281 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     2341 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     2401 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2461 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     2521 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     2581 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     2641 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     2701 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     2761 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     2821 gcagagctct ccctatcagt gatagagatc tccctatcag tgatagagat cgtcgacgag
     2881 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata
     2941 gaagacaccg ggaccgatcc agcctccggc tagagcggcc gccatgggat ccggttctgc
     3001 tagcggttca ggttctaccg gtggatcatc tagaactagc ggaagtggat ctggatcagg
     3061 aagtggttct ggatcaggta gtggttctgg tagtggatca ccatctcgcc tggaagagga
     3121 actgcgtcgt cgcctgaccg aaccaaccgg aggaagcgga gctactaact tcagcctgct
     3181 gaagcaggct ggagacgtgg aggagaaccc tggacctgga acgggcggag ctggttcaac
     3241 gtctatgagc gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt
     3301 ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca
     3361 gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc
     3421 tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt
     3481 cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg
     3541 gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt
     3601 caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg
     3661 ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga
     3721 catggccctg aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag
     3781 atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact
     3841 ggaaagaatc aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc
     3901 cagatactgc gacctcccta gcaaactggg gcacaagctt aattaactcg agtaatcaac
     3961 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     4021 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     4081 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     4141 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     4201 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     4261 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     4321 ctgacaattc cgtggtgttg ttggggaaat catcgtcctt tccttggctg ctcgcctgtg
     4381 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     4441 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     4501 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcctggtacc tttaagacca
     4561 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     4621 gggctaattc actcccaacg aagacaagat cacctgcagg acaggcgcgc cctgcttttt
     4681 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     4741 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     4801 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     4861 atctctagca cccgggcgat taaggaaagg gctagatcat tcttgaagac gaaagggcct
     4921 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     4981 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5041 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5101 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5161 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5221 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5281 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5341 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5401 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5461 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5521 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5581 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5641 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5701 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5761 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5821 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5881 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5941 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6001 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6061 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6121 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6181 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6241 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6301 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6361 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6421 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6481 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6541 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6601 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6661 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6721 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     6781 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     6841 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     6901 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     6961 cagcaagctc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     7021 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     7081 ctccccgtgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     7141 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     7201 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg acatgattac
     7261 gaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     7321 caatgtatct tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa
     7381 acttcccacc ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac
     7441 ctgtgattcc tctgaattat tttcatttta aagaaattgt atttgt
//
