LOCUS       sequence-220601-        9194 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9194)
  AUTHORS   Elegheert J, Behiels E, Bishop B, Scott S, Woolley RE, Griffiths SC,
            Byrne EFX, Chang VT, Stuart DI, Jones EY, Siebold C, Aricescu AR
  TITLE     Lentiviral transduction of mammalian cells for fast, scalable and 
            high-level production of soluble and membrane proteins.
  JOURNAL   Nat Protoc. 2018 Nov 19. pii: 10.1038/s41596-018-0075-9. doi: 
            10.1038/s41596-018-0075-9.
  PUBMED    30455477
REFERENCE   2  (bases 1 to 9194)
  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"; journalName: "Nat Protoc.
            2018 Nov 19. pii: 10.1038/s41596-018-0075-9. doi: 
            10.1038/s41596-018-0075-9."
COMMENT     Sequence Label: pHR-SFFV_3C-Twin-Strep
FEATURES             Location/Qualifiers
     source          1..9194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(21..39)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(22..39)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(329..348)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(562..581)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     681..703
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(741..759)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        827..931
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(932..1000,1001..1792)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1150..1169)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1963..2551
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2452..2471
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2705..2722
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2792..2812)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2797..3126
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2977..3112
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          4260..4325
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4455..4475
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4900..5034
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4937..4956)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4991..5010
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5202..5835
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5882..6007
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6504..6737
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6922..6966
                     /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    7251..7368
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        7527..7934
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      7953..7962
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             8070..8093
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(8106..8111,8112..8135,8136..8171,8172..8195)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     misc_feature    8259..8847
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8312..8332)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8730..8741)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8957..9190
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 ctagaattaa ttccgtgtat tctatagtgt cacctaaatc gtatgtgtat gatacataag
       61 gttatgtatt aattgtagcc gcgttctaac gacaatatgt acaagcctaa ttgtgtagca
      121 tctggcttac tgaagcagac cctatcatct ctctcgtaaa ctgccgtcag agtcggtttg
      181 gttggacgaa ccttctgagt ttctggtaac gccgtcccgc acccggaaat ggtcagcgaa
      241 ccaatcagca gggtcatcgc tagccagatc ctctacgccg gacgcatcgt ggccggcatc
      301 accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat
      361 cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc
      421 gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg
      481 ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag
      541 cgtcgaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
      601 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
      661 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
      721 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
      781 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
      841 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
      901 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
      961 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     1021 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     1081 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     1141 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     1201 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     1261 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     1321 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     1381 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     1441 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     1501 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     1561 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     1621 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     1681 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     1741 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     1801 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     1861 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     1921 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     1981 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2041 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2101 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2161 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2221 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2281 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2341 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     2401 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2461 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     2521 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     2581 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     2641 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     2701 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     2761 cgcgcgttgg ccgattcatt aatgcagctg tggaatgtgt gtcagttagg gtgtggaaag
     2821 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2881 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2941 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3001 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3061 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3121 tgcaaaaagc ttggacacaa gacaggcttg cgagatatgt ttgagaatac cactttatcc
     3181 cgcgtcaggg agaggcagtg cgtaaaaaga cgcggactca tgtgaaatac tggtttttag
     3241 tgcgccagat ctctataatc tcgcgcaacc tattttcccc tcgaacactt tttaagccgt
     3301 agataaacag gctgggacac ttcacatgag cgaaaaatac atcgtcacct gggacatgtt
     3361 gcagatccat gcacgtaaac tcgcaagccg actgatgcct tctgaacaat ggaaaggcat
     3421 tattgccgta agccgtggcg gtctgtaccg ggtgcgttac tggcgcgtga actgggtatt
     3481 cgtcatgtcg ataccgtttg tatttccagc tacgatcacg acaaccagcg cgagcttaaa
     3541 gtgctgaaac gcgcagaagg cgatggcgaa ggcttcatcg ttattgatga cctggtggat
     3601 tccggtggta ctgcggttgc gattcgtgaa atgtatccaa aagcgcactt tgtcaccatc
     3661 ttcgcaaaac cggctggtcg tccgctggtt gatgactatg ttgttgatat cccgcaagat
     3721 acctggattg aacagccgtg ggatatgggc gtcgtattcg tcccgccaat ctccggtcgc
     3781 taatcttttc aacgcctggc actgccgggc gttgttcttt ttaacttcag gcgggttaca
     3841 atagtttcca gtaagtattc tggaggctgc atccatgaca caggcaaacc tgagcgaaac
     3901 cctgttcaaa ccccgcttta aacatcctga aacctcgacg ctagtccgcc gctttaatca
     3961 cggcgcacaa ccgcctgtgc agtcggccct tgatggtaaa accatccctc actggtatcg
     4021 catgattaac cgtctgatgt ggatctggcg cggcattgac ccacgcgaaa tcctcgacgt
     4081 ccaggcacgt attgtgatga gcgatgccga acgtaccgac gatgatttat acgatacggt
     4141 gattggctac cgtggcggca actggattta tgagtgggcc ccggatcttt gtgaaggaac
     4201 cttacttctg tggtgtgaca taattggaca aactacctac agagatttaa agctctaagg
     4261 taaatataaa atttttaagt gtataatgtg ttaaactact gattctaatt gtttgtgtat
     4321 tttagattcc aacctatgga actgatgaat gggagcagtg gtggaatgcc tttaatgagg
     4381 aaaacctgtt ttgctcagaa gaaatgccat ctagtgatga tgaggctact gctgactctc
     4441 aacattctac tcctccaaaa aagaagagaa aggtagaaga ccccaaggac tttccttcag
     4501 aattgctaag ttttttgagt catgctgtgt ttagtaatag aactcttgct tgctttgcta
     4561 tttacaccac aaaggaaaaa gctgcactgc tatacaagaa aattatggaa aaatattctg
     4621 taacctttat aagtaggcat aacagttata atcataacat actgtttttt cttactccac
     4681 acaggcatag agtgtctgct attaataact atgctcaaaa attgtgtacc tttagctttt
     4741 taatttgtaa aggggttaat aaggaatatt tgatgtatag tgccttgact agagatcata
     4801 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     4861 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     4921 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4981 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcaactgg
     5041 ataactcaag ctaaccaaaa tcatcccaaa cttcccaccc cataccctat taccactgcc
     5101 aattacctgt ggtttcattt actctaaacc tgtgattcct ctgaattatt ttcattttaa
     5161 agaaattgta tttgttaaat atgtactaca aacttagtag ttggaagggc taattcactc
     5221 ccaaagaaga caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga
     5281 ttagcagaac tacacaccag ggccaggggt cagatatcca ctgacctttg gatggtgcta
     5341 caagctagta ccagttgagc cagataaggt agaagaggcc aataaaggag agaacaccag
     5401 cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag tgttagagtg
     5461 gaggtttgac agccgcctag catttcatca cgtggcccga gagctgcatc cggagtactt
     5521 caagaactgc tgatatcgag cttgctacaa gggactttcc gctggggact ttccagggag
     5581 gcgtggcctg ggcgggactg gggagtggcg agccctcaga tcctgcatat aagcagctgc
     5641 tttttgcctg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
     5701 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     5761 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     5821 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
     5881 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
     5941 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
     6001 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
     6061 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
     6121 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
     6181 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
     6241 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
     6301 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg gccgctgatc
     6361 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa
     6421 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag
     6481 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga
     6541 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt
     6601 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa
     6661 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta
     6721 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct
     6781 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc
     6841 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa
     6901 gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca
     6961 agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg
     7021 atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga
     7081 gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc
     7141 gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag atccattcga
     7201 ttagtgaacg gatctcgacg gtatcgccaa atggcagtat tcatccacaa ttttaaaaga
     7261 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
     7321 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
     7381 agggacagca gagatccagt ttggatcgat aagcttgata tcgaattgct gcagccccga
     7441 tagtcgacaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca
     7501 cctgtaggtt tggcaagcta gctgcagtaa cgccattttg caaggcatgg aaaaatacca
     7561 aaccaagaat agagaagttc agatcaaggg cgggtacatg aaaatagcta acgttgggcc
     7621 aaacaggata tctgcggtga gcagtttcgg ccccggcccg gggccaagaa cagatggtca
     7681 ccgcagtttc ggccccggcc cgaggccaag aacagatggt ccccagatat ggcccaaccc
     7741 tcagcagttt cttaagaccc atcagatgtt tccaggctcc cccaaggacc tgaaatgacc
     7801 ctgcgcctta tttgaattaa ccaatcagcc tgcttctcgc ttctgttcgc gcgcttctgc
     7861 ttcccgagct ctataaaaga gctcacaacc cctcactcgg cgcgccagtc ctccgacaga
     7921 ctgagtcgcc cgggtctaga gaattcaagc ttgccaccat gggtatcctt cccagccctg
     7981 ggatgcctgc gctgctctcc ctcgtgagcc ttctctccgt gctgctgatg ggttgcgtag
     8041 ctgaaaccgg tgcatctgga agtggtaccc ttgaggtgct gtttcaggga ccaggaggta
     8101 gtggatctgc ttggagccat ccacagttcg aaaaaggtgg aggttctggc ggtggatcag
     8161 gtggaagtgc atggtctcac cctcagtttg agaaataatg actcgaggac tcttgagtcg
     8221 agctgcaggc atgcaagctt gatatcgtac gtatcgataa tcaacctctg gattacaaaa
     8281 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     8341 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     8401 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     8461 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     8521 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     8581 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     8641 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     8701 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     8761 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     8821 ggatctccct ttgggccgcc tccccgcatc gataccgtcg tccacgaggg aattaattcg
     8881 agctccgtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     8941 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt
     9001 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     9061 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     9121 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     9181 atctctagca gcaa
//
