LOCUS       sequence-7011-ex        8595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral transfer vector. pPRIME cloning plasmid 
            with a CMV promoter controlling expression of Neomycin and 
            miR30-based shRNA targeting firefly luciferase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8595)
  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 8595)
  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-Neo-FF3
FEATURES             Location/Qualifiers
     source          1..8595
                     /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"
     enhancer        2659..2962
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2963..3166
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3212..4015
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3275..3294)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3885..3904
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     ncRNA           4066..4159
                     /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           4173..4285
                     /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        4438..4540
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4541..5200
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4607..4626)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             5332..5361
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    5484..6072
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5537..5557)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5955..5966)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6075..6091)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             6601..6781
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6843..7431)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6923..6942)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7602..8393,8394..8462))
                     /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     8225..8244
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8463..8567)
                     /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 cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag
     2641 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     2701 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     2761 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     2821 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     2881 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     2941 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     3001 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     3061 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     3121 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
     3181 tcagatccgc tagcgctacc gggcctgcag gatgggatcg gccattgaac aagatggatt
     3241 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3301 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3361 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     3421 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3481 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3541 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3601 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3661 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3721 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     3781 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     3841 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     3901 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     3961 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggc
     4021 cgcaagcctt gttaagtgct cgcttcggca gcacatatac tatgtttgaa tgaggcttca
     4081 gtactttaca gaatcgttgc ctgcacatct tggaaacact tgctgggatt acttcttcag
     4141 gttaacccaa cagaaggctc gagcaaccag aattcaaggg gctactttag gagcaattat
     4201 cttgtttact aaaactgaat accttgctat ctctttgata catttttaca aagctgaatt
     4261 aaaatggtat aaattaaatc acttttttca attggaagac taatgcgttt aaacacgcgg
     4321 cgacgcgttc gaccgaataa aacctgtgac ggaagatcac ttcgcagaat aaataaatcc
     4381 tggtgtccct gttgataccg ggaagccctg ggccaacttt tggcgaaaat gagacgttga
     4441 tcggcacgta agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt
     4501 ttttgagttg tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg
     4561 atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc
     4621 agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac
     4681 cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat
     4741 gaatgctcat ccggaattac gtatggcaat gaaagacggt gagctggtga tatgggatag
     4801 tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag
     4861 tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta
     4921 cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc
     4981 caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt
     5041 cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct
     5101 ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga
     5161 attacaacag tactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg
     5221 gtgcccttaa acgcctggtt gctacgcctg aataagtgat aataagcgga tgaatggcag
     5281 aaattcggat ctcgaccgcg tttgggcggt ggctccctgc cacgcggctc cgaacagaag
     5341 ctgatctccg aagaggatct gacatgtgtt taaacctcga cttaattaag tcgagggtcg
     5401 acggtatcga taagctcgct tcacgagatc atgtttaagg gttccggttc cactaggtac
     5461 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
     5521 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     5581 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     5641 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     5701 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     5761 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     5821 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     5881 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     5941 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     6001 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     6061 ccgcctcccc gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc
     6121 acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag
     6181 gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca
     6241 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     6301 caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat
     6361 tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac
     6421 aagctagtac cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc
     6481 ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg
     6541 aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact
     6601 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     6661 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     6721 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     6781 agcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6841 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6901 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6961 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     7021 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     7081 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     7141 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     7201 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     7261 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     7321 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     7381 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     7441 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     7501 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     7561 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     7621 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     7681 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     7741 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7801 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7861 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7921 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7981 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     8041 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     8101 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     8161 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     8221 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     8281 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     8341 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     8401 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     8461 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     8521 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     8581 aaagtgccac ctgac
//
