LOCUS       sequence-318316-        8747 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral transfer vector. pPRIME cloning plasmid 
            with a CMV promoter controlling expression of LNGFR and miR30-based 
            shRNA targeting firefly luciferase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8747)
  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 8747)
  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-CMV-LNGFR-FF3
FEATURES             Location/Qualifiers
     source          1..8747
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     ncRNA           1432..1553
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           1580..1594
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           1622..1748
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     promoter        1902..2004
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             2005..2664
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2071..2090)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2794..2823
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     2848..2864
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    2939..3527
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2992..3012)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3410..3421)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3530..3546)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             4056..4236
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4298..4886)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4378..4397)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5057..5848,5849..5917))
                     /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     5680..5699
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5918..6022)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6097..6116)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6288..6667
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6669..6867
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             6885..7065
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7112..7237
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7734..7967
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8152..8196
                     /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    8494..8611
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactaccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccgggcctgc aggcaccatg ggggcaggtg ccaccggccg cgccatggac gggccgcgcc
      601 tgctgctgtt gctgcttctg ggggtgtccc ttggaggtgc caaggaggca tgccccacag
      661 gcctgtacac acacagcggt gagtgctgca aagcctgcaa cctgggcgag ggtgtggccc
      721 agccttgtgg agccaaccag accgtgtgtg agccctgcct ggacagcgtg acgttctccg
      781 acgtggtgag cgcgaccgag ccgtgcaagc cgtgcaccga gtgcgtgggg ctccagagca
      841 tgtcggcgcc gtgcgtggag gccgacgacg ccgtgtgccg ctgcgcctac ggctactacc
      901 aggatgagac gactgggcgc tgcgaggcgt gccgcgtgtg cgaggcgggc tcgggcctcg
      961 tgttctcctg ccaggacaag cagaacaccg tgtgcgagga gtgccccgac ggcacgtatt
     1021 ccgacgaggc caaccacgtg gacccgtgcc tgccctgcac cgtgtgcgag gacaccgagc
     1081 gccagctccg cgagtgcaca cgctgggccg acgccgagtg cgaggagatc cctggccgtt
     1141 ggattacacg gtccacaccc ccagagggct cggacagcac agcccccagc acccaggagc
     1201 ctgaggcacc tccagaacaa gacctcatag ccagcacggt ggcaggtgtg gtgaccacag
     1261 tgatgggcag ctcccagccc gtggtgaccc gaggcaccac cgacaacctc atccctgtct
     1321 attgctccat cctggctgct gtggttgtgg gtcttgtggc ctacatagcc ttcaagaggt
     1381 gagcggccgc aagccttgtt aagtgctcgc ttcggcagca catatactat gttgaatgag
     1441 gcttcagtac tttacagaat cgttgcctgc acatcttgga aacacttgct gggattactt
     1501 cttcaggtta acccaacaga aggctcgaga aggtatattg ctgttgacag tgagcgagct
     1561 cccgtgaatt ggaatcctag tgaagccaca gatgtaggat tccaattcag cgggagcctg
     1621 cctactgcct cggaattcaa ggggctactt taggagcaat tatcttgttt actaaaactg
     1681 aataccttgc tatctctttg atacattttt acaaagctga attaaaatgg tataaattaa
     1741 atcacttttt tcaattggaa gactaatgcg tttaaacacg cggcgacgcg ttcgaccgaa
     1801 taaatacctg tgacggaaga tcacttcgca gaataaataa atcctggtgt ccctgttgat
     1861 accgggaagc cctgggccaa cttttggcga aaatgagacg ttgatcggca cgtaagaggt
     1921 tccaactttc accataatga aataagatca ctaccgggcg tattttttga gttgtcgaga
     1981 ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat
     2041 atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc
     2101 tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag
     2161 cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa
     2221 ttacgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac
     2281 accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat
     2341 ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc
     2401 tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt
     2461 ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc
     2521 atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat
     2581 catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc
     2641 gatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc ttaaacgcct
     2701 ggttgctacg cctgaataag tgataataag cggatgaatg gcagaaattc ggatctcgcg
     2761 cgtttgggcg gtggctccct gccacgcggc tccgaacaga agctgatctc cgaagaggat
     2821 ctgacatgtg tttaaactta attaagtcga ggtcgacggt atcgataagc tcgcttcacg
     2881 agatcatgtt taagggttcc ggttccacta ggtacaattc gatatcaagc ttatcgataa
     2941 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3001 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3061 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     3121 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     3181 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     3241 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     3301 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     3361 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     3421 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     3481 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg
     3541 acctcgatcg agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca
     3601 atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac
     3661 ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     3721 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc
     3781 tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag
     3841 ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga
     3901 aggtagaaga agccaatgaa ggagagaaca cccgcttgtt acaccctgtg agcctgcatg
     3961 ggatggatga cccggagaga gaagtattag agtggaggtt tgacagccgc ctagcatttc
     4021 atcacatggc ccgagagctg catccggact gtactgggtc tctctggtta gaccagatct
     4081 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     4141 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     4201 tcagaccctt ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa
     4261 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4321 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4381 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4441 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4501 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4561 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4621 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4681 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4741 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4801 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4861 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4921 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4981 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5041 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5101 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5161 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5221 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5281 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5341 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5401 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5461 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5521 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5581 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5641 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5701 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5761 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5821 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5881 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5941 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6001 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     6061 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     6121 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     6181 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     6241 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
     6301 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     6361 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     6421 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     6481 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     6541 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     6601 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     6661 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     6721 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     6781 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     6841 tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct ctctggttag
     6901 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     6961 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     7021 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag
     7081 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga
     7141 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
     7201 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
     7261 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
     7321 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
     7381 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
     7441 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
     7501 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
     7561 caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     7621 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     7681 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     7741 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     7801 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     7861 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     7921 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     7981 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     8041 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     8101 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     8161 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     8221 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     8281 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     8341 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     8401 ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact gcgtgcgcca
     8461 attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     8521 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     8581 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     8641 agtttggtta gtaccgggcc cgctctagac gtattaccgc catgcattag ttattaatag
     8701 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccg
//
