LOCUS       sequence-9888-ex        7807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd gen lentiviral Luciferase shRNA (H1/TO promoter), 3'LTR 
            insertion, Puro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7807)
  AUTHORS   Campeau E, Ruhl VE, Rodier F, Smith CL, Rahmberg BL, Fuss JO, 
            Campisi J, Yaswen P, Cooper PK, Kaufman PD.
  TITLE     A versatile viral system for expression and depletion of proteins in
            mammalian cells.
  JOURNAL   PLoS One. 2009 Aug 6;4(8):e6529.
  PUBMED    19657394
REFERENCE   2  (bases 1 to 7807)
  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: "PLoS One.";
            date: "2009-08-6"; volume: "4(8)"; pages: "e6529"
COMMENT     Sequence Label: pLenti X2 Puro/pTER shLUC (w589-1)
FEATURES             Location/Qualifiers
     source          1..7807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        27..131
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(132..200,201..992)
                     /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(350..369)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1163..1751
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1652..1671
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1905..1922
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2039..2060
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2075..2080,2081..2098,2099..2105)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2113..2129
                     /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     2118..2140
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2137..2153
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2137..2153
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2172..2192
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2174..2192
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     LTR             2448..2628
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2672..2797
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3290..3523
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3707..3751
                     /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    4012..4129
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_feature    4183..4771
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4236..4256)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4654..4665)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        4785..5284
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4820..4843)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5217..5236
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             5305..5904
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5305..5324)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5801..5821
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    6085..6109
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    6242..6260
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(6306..6325)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    complement(6502..6526)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     LTR             6601..6781
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    6853..6974
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6890..6909)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6944..6963
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6993..7128
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7160..7179)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7161..7179)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7189..7206)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7189..7205)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7198..7220)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7350..7805
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7437..7456)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7647..7668
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
       61 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
      121 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
      181 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
      241 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
      301 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
      361 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
      421 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
      481 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
      541 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
      601 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
      661 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
      721 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
      781 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
      841 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
      901 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
      961 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     1021 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     1081 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     1141 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     1201 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     1261 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     1321 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     1381 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     1441 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     1501 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     1561 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     1621 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     1681 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     1741 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     1801 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     1861 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     1921 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     1981 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     2041 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     2101 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     2161 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat
     2221 gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc
     2281 cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg
     2341 tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc
     2401 gcattgcaga gatattgtat ttaagtgcct agctcgatac aataaacggg tctctctggt
     2461 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     2521 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     2581 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
     2641 cagggacctg aaagcgaaag ggaaaccaga gctctctcga cgcaggactc ggcttgctga
     2701 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
     2761 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
     2821 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
     2881 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
     2941 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
     3001 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
     3061 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
     3121 caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt
     3181 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca
     3241 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt
     3301 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcctcaatg acgctgacgg
     3361 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta
     3421 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa
     3481 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct
     3541 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc
     3601 tggaacagat tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     3661 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     3721 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     3781 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     3841 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     3901 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
     3961 gagagacaga gacagatcca ttcgattagt gaacggatct cgacggttaa cttttaaaag
     4021 aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga
     4081 catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaatttta ttccagtgtg
     4141 gtggaattct gcagatatcc agcacagtgg cggccgctcg acaatcaacc tctggattac
     4201 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     4261 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     4321 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     4381 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     4441 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     4501 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     4561 gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg
     4621 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     4681 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     4741 agtcggatct ccctttgggc cgcctccccg cctggaattc taccgggtag gggaggcgct
     4801 tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac
     4861 aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac cggctccgtt
     4921 ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc
     4981 cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt
     5041 gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata
     5101 gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg
     5161 gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt
     5221 ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct
     5281 ttcgacctgc agcccaagct taccatgacc gagtacaagc ccacggtgcg cctcgccacc
     5341 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     5401 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     5461 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     5521 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     5581 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     5641 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     5701 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     5761 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     5821 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     5881 atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg
     5941 agcgcacgac cccatgcatc tcgagggccc ggtaccttta agaccaatga cttacaaggc
     6001 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc tagactcgag
     6061 cggccgccac tgtgctggat atcaaccact ttgtacaaga aagctgggtc tagatatctc
     6121 gagtgcggcc gcgaattcca ccacactgga ctagtggatc cgagctcggt accaagcttt
     6181 tccaaaaact tacgctgagt acttcgagga cagcacactc gaagtactca gcgtaagggg
     6241 atctctatca ctgataggga acttataaga ttcccaaatc caaagacatt tcacgtttat
     6301 ggtgatttcc cagaacacat agcgacatgc aaatattgca gggcgccact cccctgtccc
     6361 tcacagccat cttcctgcca gggcgcacgc gcgctgggtg ttcccgccta gtgacactgg
     6421 gccgcgattc cttggagcgg gttgatgacg tcagcgttcg aattcggtac cggatccagt
     6481 cgactgaatt ggttccttta aagcctgctt ttttgtacaa acttgttgat atctgcagaa
     6541 ttccaccaca ctggactagc tcactcccaa cgaagacaag atctgctttt tgcttgtact
     6601 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctgcct agggaaccca
     6661 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     6721 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     6781 agtagtagtt catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca
     6841 gagagtgaga ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     6901 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     6961 atcaatgtat cttatcatgt ctggctctag ctatcccgcc cctaactccg cccagttccg
     7021 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     7081 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgcg
     7141 tcgagacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     7201 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     7261 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     7321 gttgcgcagc ctgaatggcg aatggcgcga cgcgccctgt agcggcgcat taagcgcggc
     7381 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     7441 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     7501 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     7561 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     7621 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     7681 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     7741 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     7801 aatttcc
//
