LOCUS       sequence-389898-        7375 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a N-terminally GFP-3C-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7375)
  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 7375)
  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: pTS94 pHAGE2 CMVtetO2 GFP-22xGS-3C-MCS
FEATURES             Location/Qualifiers
     source          1..7375
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    677..802
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1299..1532
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1717..1761
                     /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    2054..2171
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2215..2594
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2595..2798
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2749..2769
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2800..2818
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2821..2839
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2849..2873
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2948..2957
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3719..3742
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     misc_feature    3813..4401
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3866..3886)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4284..4295)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4549..4729
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4776..4794)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4862..4966
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4967..5035,5036..5827)
                     /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(5185..5204)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5998..6586
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6740..6757
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6997..7018
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7033..7038,7039..7056,7057..7063)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7071..7087
                     /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     7076..7098
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7095..7111
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7095..7111
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7161..7180
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(7372..7375,1..630)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 agggctaatt cactcccaaa gaagacaaga tatccttgat ctgtggatct accacacaca
       61 aggctacttc cctgattagc agaactacac accagggcca ggggtcagat atccactgac
      121 ctttggatgg tgctacaagc tagtaccagt tgagccagat aaggtagaag aggccaataa
      181 aggagagaac accagcttgt tacaccctgt gagcctgcat gggatggatg acccggagag
      241 agaagtgtta gagtggaggt ttgacagccg cctagcattt catcacgtgg cccgagagct
      301 gcatccggag tacttcaaga actgctgata tcgagcttgc tacaagggac tttccgctgg
      361 ggactttcca gggaggcgtg gcctgggcgg gactggggag tggcgagccc tcagatcctg
      421 catataagca gctgcttttt gcctgtactg ggtctctctg gttagaccag atctgagcct
      481 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
      541 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
      601 ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa
      661 agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag
      721 aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga
      781 gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa
      841 aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag
      901 cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag
      961 acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt
     1021 atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa
     1081 ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc
     1141 ggccggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa
     1201 ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag
     1261 agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc
     1321 ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga
     1381 caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa
     1441 cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct
     1501 gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc
     1561 atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt
     1621 tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata
     1681 cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa
     1741 ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata
     1801 aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt
     1861 tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca
     1921 accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga
     1981 gacagatcca ttcgattagt gaacggatct cgacggtatc gccgaattca caaatggcag
     2041 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     2101 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     2161 ttcaaaattt tcgggtttat tacagggaca gcagagatcc agtttggacg cgttgacatt
     2221 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     2281 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     2341 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     2401 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     2461 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     2521 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     2581 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     2641 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     2701 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     2761 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
     2821 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
     2881 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccggc
     2941 tagagcggcc gccatggtgt ctaaaggtga agaacttttt acgggtgtcg tccccatcct
     3001 ggttgaattg gacggagacg tgaatggcca taaattttca gtgagcggtg aaggagaggg
     3061 agacgccact tacggtaaac ttaccttgaa atttatctgc actaccggga aactgcccgt
     3121 accctggccg acgctcgtaa caacgcttac ctacggcgtg caatgtttta gcagataccc
     3181 agaccatatg aagcagcacg attttttcaa gagtgcaatg cctgaggggt acgtacagga
     3241 aaggacgatc tttttcaaag acgatggtaa ttataaaacg cgggctgagg ttaaatttga
     3301 aggagacacg ctcgtgaatc gaatagagct gaagggaatc gacttcaagg aagacggtaa
     3361 catcctgggc cataaactcg aatataatta caattcacac aacgtctata tcatggcaga
     3421 taaacagaag aatggcatca aagtcaattt taagatccgg cataatatcg aggacggatc
     3481 agtacagctt gccgaccatt accaacagaa tacaccgata ggagacggtc ccgtcctctt
     3541 gccagataat cactatttga gcacacaatc cgctctttct aaagatccga atgaaaagcg
     3601 cgatcatatg gtcttgaaag agttcgtcac tgccgccggc ataaccggat ctggcagtgg
     3661 atcaggatct ggtagtggtt ctggtagcgg atctggaagt ggcagtggat ctggtagctt
     3721 agaagttttg tttcaaggtc caggatcttc tagtggatcc ggttctgcta gcggttcagg
     3781 ttctaccggt ggatcatcta gataactcga gtaatcaacc tctggattac aaaatttgtg
     3841 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     3901 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     3961 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     4021 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     4081 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     4141 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     4201 tggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     4261 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     4321 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     4381 ccctttgggc cgcctccccg cctggtacct ttaagaccaa tgacttacaa ggcagctgta
     4441 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     4501 agacaagatc acctgcagga caggcgcgcc ctgctttttg cttgtactgg gtctctctgg
     4561 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     4621 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     4681 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcac ccgggcgatt
     4741 aaggaaaggg ctagatcatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
     4801 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     4861 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4921 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4981 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5041 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5101 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5161 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     5221 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5281 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5341 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5401 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5461 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5521 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5581 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5641 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5701 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5761 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5821 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5881 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5941 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6001 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6061 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6121 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6181 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6241 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6301 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6361 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6421 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6481 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6541 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6601 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     6661 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     6721 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     6781 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agcaagctca tggctgacta
     6841 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     6901 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tccccgtggc acgacaggtt
     6961 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
     7021 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     7081 ataacaattt cacacaggaa acagctatga catgattacg aatttcacaa ataaagcatt
     7141 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     7201 gatcaactgg ataactcaag ctaaccaaaa tcatcccaaa cttcccaccc cataccctat
     7261 taccactgcc aattacctgt ggtttcattt actctaaacc tgtgattcct ctgaattatt
     7321 ttcattttaa agaaattgta tttgttaaat atgtactaca aacttagtag ttgga
//
