LOCUS       sequence-376120-        7313 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector to generate TwinStrep-tagged TMPRSS2 stable 
            expressing cell line.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7313)
  AUTHORS   Richards CM, Jabs S, Qiao W, Varanese LD, Schweizer M, Mosen PR, 
            Riley NM, Klussendorf M, Zengel JR, Flynn RA, Rustagi A, Widen JC, 
            Peters CE, Ooi YS, Xie X, Shi PY, Bartenschlager R, Puschnik AS, 
            Bogyo M, Bertozzi CR, Blish CA, Winter D, Nagamine CM, Braulke T, 
            Carette JE
  TITLE     The human disease gene LYSET is essential for lysosomal enzyme 
            transport and viral infection.
  JOURNAL   Science. 2022 Sep 8:eabn5648. doi: 10.1126/science.abn5648.
  PUBMED    36074821
REFERENCE   2  (bases 1 to 7313)
  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: "Science. 
            2022 Sep 8:eabn5648. doi: 10.1126/science.abn5648."
COMMENT     Sequence Label: pLenti-CMV-TMPRSS2-TwinStrep_Zeo
FEATURES             Location/Qualifiers
     source          1..7313
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        114..161
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             180..554
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     LTR             647..880
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    952..1073
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(989..1008)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1043..1062
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1268..1856
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1757..1776
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2010..2027
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2144..2165
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2180..2185,2186..2203,2204..2210)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2218..2234
                     /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     2223..2245
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2242..2258
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2242..2258
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2277..2297
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2279..2297
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2325..2551
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             2552..2732
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2779..2904
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3397..3630
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3815..3859
                     /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    4126..4242
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        4265..4568
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4569..4772
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4723..4743
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4769..4793
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             6341..6364
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             6401..6424
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     misc_feature    6442..7030
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6495..6515)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6913..6924)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7081..7101)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(7086..7313,1..102)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(7266..7313,1..88)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
       61 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc cctgttgaca
      121 attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca
      181 tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg
      241 agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg
      301 tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca
      361 acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg
      421 tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc
      481 cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg
      541 aggagcagga ctgacacgtg ctacgagatt taaatggtac ctttaagacc aatgacttac
      601 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctagct
      661 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
      721 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
      781 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
      841 tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt
      901 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt
      961 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     1021 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     1081 tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     1141 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcca
     1201 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     1261 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     1321 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     1381 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     1441 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     1501 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     1561 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     1621 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     1681 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     1741 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     1801 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     1861 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     1921 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     1981 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     2041 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     2101 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     2161 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     2221 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa
     2281 ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt cttatgcaat
     2341 actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac aaggagagaa
     2401 aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct tattaggaag
     2461 gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt gcagagatat
     2521 tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc agatctgagc
     2581 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
     2641 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
     2701 acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg
     2761 aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca
     2821 agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag
     2881 gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa
     2941 aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca
     3001 agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt
     3061 agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca
     3121 ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc
     3181 aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa
     3241 gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt
     3301 atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag
     3361 aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt
     3421 gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca
     3481 attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca
     3541 gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt
     3601 ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat
     3661 ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg
     3721 gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca
     3781 ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt
     3841 agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa
     3901 attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc
     3961 tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc acctcccaac
     4021 cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gagacagaga
     4081 cagatccatt cgattagtga acggatctcg acggtatcgg ttaactttta aaagaaaagg
     4141 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     4201 aactaaagaa ttacaaaaac aaattacaaa attcaaaatt ttatcgataa gcttgggagt
     4261 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     4321 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     4381 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     4441 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     4501 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     4561 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     4621 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     4681 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     4741 gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga
     4801 gacgccatcc acgctgtttt gacctccata gaagacaccg actctagaga ccatggcttt
     4861 gaactcaggg tcaccaccag ctattggacc ttactatgaa aaccatggat accaaccgga
     4921 aaacccctat cccgcacagc ccactgtggt ccccactgtc tacgaggtgc atccggctca
     4981 gtactacccg tcccccgtgc cccagtacgc cccgagggtc ctgacgcagg cttccaaccc
     5041 cgtcgtctgc acgcagccca aatccccatc cgggacagtg tgcacctcaa agactaagaa
     5101 agcactgtgc atcaccttga ccctggggac cttcctcgtg ggagctgcgc tggccgctgg
     5161 cctactctgg aagttcatgg gcagcaagtg ctccaactct gggatagagt gcgactcctc
     5221 aggtacctgc atcaacccct ctaactggtg tgatggcgtg tcacactgcc ccggcgggga
     5281 ggacgagaat cggtgtgttc gcctctacgg accaaacttc atccttcagg tgtactcatc
     5341 tcagaggaag tcctggcacc ctgtgtgcca agacgactgg aacgagaact acgggcgggc
     5401 ggcctgcagg gacatgggct ataagaataa tttttactct agccaaggaa tagtggatga
     5461 cagcggatcc accagcttta tgaaactgaa cacaagtgcc ggcaatgtcg atatctataa
     5521 aaaactgtac cacagtgatg cctgttcttc aaaagcagtg gtttctttac gctgtatagc
     5581 ctgcggggtc aacttgaact caagccgcca gagcaggatc gtgggcggcg agagcgcgct
     5641 cccgggggcc tggccctggc aggtcagcct gcacgtccag aacgtccacg tgtgcggagg
     5701 ctccatcatc acccccgagt ggatcgtgac agccgcccac tgcgtggaaa aacctcttaa
     5761 caatccatgg cattggacgg catttgcggg gattttgaga caatctttca tgttctatgg
     5821 agccggatac caagtagaaa aagtgatttc tcatccaaat tatgactcca agaccaagaa
     5881 caatgacatt gcgctgatga agctgcagaa gcctctgact ttcaacgacc tagtgaaacc
     5941 agtgtgtctg cccaacccag gcatgatgct gcagccagaa cagctctgct ggatttccgg
     6001 gtggggggcc accgaggaga aagggaagac ctcagaagtg ctgaacgctg ccaaggtgct
     6061 tctcattgag acacagagat gcaacagcag atatgtctat gacaacctga tcacaccagc
     6121 catgatctgt gccggcttcc tgcaggggaa cgtcgattct tgccagggtg acagtggagg
     6181 gcctctggtc acttcgaaga acaatatctg gtggctgata ggggatacaa gctggggttc
     6241 tggctgtgcc aaagcttaca gaccaggagt gtacgggaat gtgatggtat tcacggactg
     6301 gatttatcga caaatgaggg cagacggcgg aggcggtgga tggagccacc cccagttcga
     6361 gaagggcggc ggcagcggcg gcggcagcgg cggcggcagc tggagccacc cccagttcga
     6421 gaaggggccc gtttaatcga caatcaacct ctggattaca aaatttgtga aagattgact
     6481 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     6541 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     6601 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     6661 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     6721 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     6781 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg
     6841 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     6901 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     6961 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     7021 gcctccccgc ctggaattct gcagatatcc ggttagtaat gagtttggaa ttaattctgt
     7081 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     7141 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     7201 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     7261 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggc
//
