LOCUS       sequence-389899-        7627 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Lentiviral transfer plasmid for doxycycline inducible expression of 
            a N-terminally GFP-SUMOEu-tagged protein in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7627)
  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 7627)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pTS96 pHAGE2 CMVtetO2 GFP-22xGS-SUMOEu-MCS
FEATURES             Location/Qualifiers
     source          1..7627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    667..792
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1289..1522
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1707..1751
                     /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    2044..2161
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2205..2584
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2585..2788
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2739..2759
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2790..2808
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2811..2829
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2839..2863
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2938..2947
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    4055..4643
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4108..4128)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4526..4537)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4791..4971
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5018..5036)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5104..5208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5209..5277,5278..6069)
                     /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(5427..5446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6240..6828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6982..6999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7239..7260
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7275..7280,7281..7298,7299..7305)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7313..7329
                     /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     7318..7340
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7337..7353
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7337..7353
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7403..7422
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(7614..7627,1..620)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 cactcccaaa gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc
       61 cctgattagc agaactacac accagggcca ggggtcagat atccactgac ctttggatgg
      121 tgctacaagc tagtaccagt tgagccagat aaggtagaag aggccaataa aggagagaac
      181 accagcttgt tacaccctgt gagcctgcat gggatggatg acccggagag agaagtgtta
      241 gagtggaggt ttgacagccg cctagcattt catcacgtgg cccgagagct gcatccggag
      301 tacttcaaga actgctgata tcgagcttgc tacaagggac tttccgctgg ggactttcca
      361 gggaggcgtg gcctgggcgg gactggggag tggcgagccc tcagatcctg catataagca
      421 gctgcttttt gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
      481 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
      541 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
      601 agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca
      661 gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg
      721 cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg
      781 tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta
      841 aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta
      901 gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg
      961 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
     1021 gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta
     1081 gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccggccgc
     1141 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
     1201 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
     1261 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
     1321 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
     1381 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
     1441 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
     1501 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
     1561 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
     1621 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
     1681 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat
     1741 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
     1801 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga
     1861 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
     1921 gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca
     1981 ttcgattagt gaacggatct cgacggtatc gccgaattca caaatggcag tattcatcca
     2041 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     2101 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     2161 tcgggtttat tacagggaca gcagagatcc agtttggacg cgttgacatt gattattgac
     2221 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     2281 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     2341 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     2401 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     2461 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     2521 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     2581 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     2641 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     2701 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     2761 acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc tccctatcag
     2821 tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc
     2881 acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccggc tagagcggcc
     2941 gccatggtgt ctaaaggtga agaacttttt acgggtgtcg tccccatcct ggttgaattg
     3001 gacggagacg tgaatggcca taaattttca gtgagcggtg aaggagaggg agacgccact
     3061 tacggtaaac ttaccttgaa atttatctgc actaccggga aactgcccgt accctggccg
     3121 acgctcgtaa caacgcttac ctacggcgtg caatgtttta gcagataccc agaccatatg
     3181 aagcagcacg attttttcaa gagtgcaatg cctgaggggt acgtacagga aaggacgatc
     3241 tttttcaaag acgatggtaa ttataaaacg cgggctgagg ttaaatttga aggagacacg
     3301 ctcgtgaatc gaatagagct gaagggaatc gacttcaagg aagacggtaa catcctgggc
     3361 cataaactcg aatataatta caattcacac aacgtctata tcatggcaga taaacagaag
     3421 aatggcatca aagtcaattt taagatccgg cataatatcg aggacggatc agtacagctt
     3481 gccgaccatt accaacagaa tacaccgata ggagacggtc ccgtcctctt gccagataat
     3541 cactatttga gcacacaatc cgctctttct aaagatccga atgaaaagcg cgatcatatg
     3601 gtcttgaaag agttcgtcac tgccgccggc ataaccggat ctggcagtgg atcaggatct
     3661 ggtagtggtt ctggtagcgg atctggaagt ggcagtggat cttctgctgc gggtggcgaa
     3721 gaagataaga aaccggcagg tggcgaaggt ggcggtgccc atatcaacct gaaagtgaaa
     3781 ggtcaagacg gcaacgaagt ctttttccgc atcaaacgtt ctacccagct gaaaaagctg
     3841 atgaacgcat actgtgaccg tcagtctgta gacatgaagg caattgcttt cctctttaag
     3901 ggtcgtcgcc tacgtgcgga aaggaccccg gatgaactgg aaatggaaga tggcgacgaa
     3961 atcgacgcaa tgctgcatca gactggtggc tctagtggat ccggttctgc tagcggttca
     4021 ggttctaccg gtggatcatc tagataactc gagtaatcaa cctctggatt acaaaatttg
     4081 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     4141 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     4201 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     4261 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     4321 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     4381 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     4441 gttggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
     4501 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     4561 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     4621 ctccctttgg gccgcctccc cgcctggtac ctttaagacc aatgacttac aaggcagctg
     4681 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
     4741 gaagacaaga tcacctgcag gacaggcgcg ccctgctttt tgcttgtact gggtctctct
     4801 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     4861 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     4921 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc acccgggcga
     4981 ttaaggaaag ggctagatca ttcttgaaga cgaaagggcc tcgtgatacg cctattttta
     5041 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     5101 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5161 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5221 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5281 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5341 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5401 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc
     5461 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5521 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5581 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5641 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5701 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5761 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5821 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5881 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5941 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     6001 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6061 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6121 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6181 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6241 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6301 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6361 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6421 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6481 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6541 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6601 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6661 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6721 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6781 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6841 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6901 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6961 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     7021 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagcaagct catggctgac
     7081 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     7141 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctccccgtg gcacgacagg
     7201 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     7261 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     7321 ggataacaat ttcacacagg aaacagctat gacatgatta cgaatttcac aaataaagca
     7381 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     7441 tggatcaact ggataactca agctaaccaa aatcatccca aacttcccac cccataccct
     7501 attaccactg ccaattacct gtggtttcat ttactctaaa cctgtgattc ctctgaatta
     7561 ttttcatttt aaagaaattg tatttgttaa atatgtacta caaacttagt agttggaagg
     7621 gctaatt
//
