LOCUS       sequence-7001-ex        8350 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral transfer vector. pPRIME-mating recipient 
            plasmid with a tetracycline-responsive promoter (TET) controlling 
            GFP expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8350)
  AUTHORS   Stegmeier F, Hu G, Rickles RJ, Hannon GJ, Elledge SJ
  TITLE     A lentiviral microRNA-based system for single-copy polymerase 
            II-regulated RNA interference in mammalian cells.
  JOURNAL   Proc Natl Acad Sci U S A. 2005 Sep 13. 102(37):13212-7.
  PUBMED    16141338
REFERENCE   2  (bases 1 to 8350)
  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: "Proc Natl 
            Acad Sci U S A. 2005 Sep 13. 102(37):13212-7."
COMMENT     Sequence Label: pPRIME-TET-GFP-recipient
FEATURES             Location/Qualifiers
     source          1..8350
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    1023..1148
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1645..1878
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2063..2107
                     /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    2405..2522
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2597..2911
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    2605..2623
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2641..2659
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2676..2694
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2712..2730
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2748..2766
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2783..2801
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2819..2837
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2884..2908
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2922..2931
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2928..2930,2931..2933,2934..3647)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2928..2930,2931..2933,2934..3644)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2973..2994)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3234..3253)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3581..3602
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5087..5116
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    5239..5827
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5292..5312)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5710..5721)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5830..5846)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             6356..6536
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6598..7186)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6678..6697)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7357..8148,8149..8217))
                     /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     7980..7999
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8218..8322)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata gaccagatct gagcctggga gctctctggc
      841 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
      901 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
      961 tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg
     1021 agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc
     1081 gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg
     1141 agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc
     1201 cagggggaaa gaaaaaatat aaataaacat atagtatggg caagcaggga gctagaacga
     1261 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
     1321 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
     1381 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     1441 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgcgctgat
     1501 cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa
     1561 agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca
     1621 gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg
     1681 aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg
     1741 tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca
     1801 actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct
     1861 aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc
     1921 tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac
     1981 ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga
     2041 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
     2101 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
     2161 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
     2221 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc
     2281 cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg
     2341 attagtgaac ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc
     2401 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
     2461 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
     2521 ttcgggttta ttacagggac agcagagatc cagtttggtt agtaccgggc ccgctctaga
     2581 cctttcgtct tcattcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttac
     2641 tccctatcag tgatagagaa cgatgtcgag tttactccct atcagtgata gagaacgtat
     2701 gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga
     2761 tagagaacgt atgtcgagtt tatccctatc agtgatagag aacgtatgtc gagtttactc
     2821 cctatcagtg atagagaacg tatgtcgagg taggcgtgta cggtgggagg cctatataag
     2881 cagagctcgt ttagtgaacc gtcagatcgc ctggaccggt cgccaccatg gtgagcaagg
     2941 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     3001 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     3061 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     3121 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     3181 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     3241 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     3301 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     3361 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     3421 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     3481 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     3541 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     3601 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtagcgg ccgcaagcct
     3661 tgttaagtgc tcgcttcggc agcacatata ctatgtttga atgaggcttc agtactttac
     3721 agaatcgttg cctgcacatc ttggaaacac ttgctgggat tacttcttca ggttaaccca
     3781 acagaaggcc atgggtctag atagggataa cagggtaatg gtaccggatc ccccaacttc
     3841 caggcccgcc atagtgattt gatttgccag cgcatcgcta tcaatcgccg ggttcaccca
     3901 ttcgcgtaac cacagttcac tgaatttcat aatctattcc tgccttattt aaactgtttg
     3961 aggaaacgca gatcgttttc gaagaatgaa cgcaggtcgg tgacgccgta acgcaacata
     4021 gtcagacgct ccatccccat cccgaagccg aaaccagagt aaacttccgg gtcgatgcca
     4081 acgttacgca acacgttcgg atgcaccatc ccgcagccca gcacttccag ccatttaccg
     4141 tttttaccca tgacgtccac ttctgcagaa ggttcggtaa acgggaagta ggaaggacgg
     4201 aagcgaatct gcaaatcttc ctcaaagaag ttacgcagga agtcgtgcag cgtgcctttc
     4261 aggttggtaa agctgatgtt ggtatcaaca atcagacctt ccatctgatg gaacatcggc
     4321 gtgtgagtct ggtcgtagtc gttacgataa acacggccag gcgcgatgat acgaatcggt
     4381 ggctgctggg ctttcatggt gcggatctgt acgccagagg tctgggtacg cagcaggcgg
     4441 gtagtgtcaa accagaaagt gtcgtggtca gcgcgcgccg ggtggtgacc aggaatgttc
     4501 agagcatcga agttatgata atcgtcttcg atttccggcc cggttgccac ggtaaagcca
     4561 agctcaccga agaaactttc gatacggtcg atggtacggg taaccggatg cagaccgccg
     4621 ttttcaatgc gacgacctgg cagagagaca tcaatcgttt ccgccgccag acgcgcattc
     4681 agtgcagcgc tttccagttc cgctttacgc gcattcagcg cctgctgaac ctgctctttc
     4741 gcttcgttga taaccgcacc agctgccgga cgctcttctg gcggcagctc acgcagggtc
     4801 gtcatctgaa gggttaagtg ccctttttta cccaaatatt cgacgcgcac attatctaac
     4861 gcggcaacat ctgacgcctg gctaatggcc gccttcgcac tggcaaccag ttctgcgaga
     4921 tgtgacatgg ttttcctcat tgtgtcagtg gtgacactgg ttcgttggac ttagagccta
     4981 tcccatcagg ctattttact tgccattttg gtccccgata ccgtcgaccg cggattaccc
     5041 tgttatccct agatctttgg gcggtggctc cctgccacgc ggctccgaac agaagctgat
     5101 ctccgaagag gatctgacat gtgtttaaac ctcgacttaa ttaagtcgag ggtcgacggt
     5161 atcgataagc tcgcttcacg agatcatgtt taagggttcc ggttccacta ggtacaattc
     5221 gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     5281 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     5341 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     5401 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     5461 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     5521 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     5581 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     5641 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     5701 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     5761 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     5821 tccccgcatc gataccgtcg acctcgatcg agacctagaa aaacatggag caatcacaag
     5881 tagcaataca gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggagga
     5941 ggtgggtttt ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgt
     6001 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg
     6061 aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattggca
     6121 gaactacaca ccagggccag ggatcagata tccactgacc tttggatggt gctacaagct
     6181 agtaccagtt gagcaagaga aggtagaaga agccaatgaa ggagagaaca cccgcttgtt
     6241 acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtattag agtggaggtt
     6301 tgacagccgc ctagcatttc atcacatggc ccgagagctg catccggact gtactgggtc
     6361 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     6421 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     6481 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagcat
     6541 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     6601 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     6661 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6721 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6781 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6841 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6901 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6961 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     7021 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     7081 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     7141 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     7201 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     7261 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     7321 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     7381 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     7441 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     7501 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     7561 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     7621 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     7681 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7741 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7801 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7861 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7921 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     7981 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     8041 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     8101 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     8161 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     8221 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     8281 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     8341 gccacctgac
//
