LOCUS       sequence-8935-ex        7885 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Retroviral eGFP shRNA #2 (H1/TO promoter), 3'LTR insertion, Neo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7885)
  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 7885)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pQCXIN X2/pTER shEGFP#1 (w491-1)
FEATURES             Location/Qualifiers
     source          1..7885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        309..512
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             513..688
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    751..950
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1151..1567
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1497..1519
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1535..1551
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     enhancer        1604..1907
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1908..2111
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2108..2132
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     misc_feature    join(2290..2863,2864..2866,2867..2875)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2457..2474)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2684..2703
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2876..3670
                     /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="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2930..2949)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3540..3559
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    4153..4177
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    4308..4326
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(4372..4391)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    complement(4568..4592)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(5119..5141)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5277..5297)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5282..5611
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5462..5597
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5633..5652
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5733..5750)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5904..6492)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5984..6003)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6663..7454,7455..7523))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7286..7305
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7524..7628)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7696..7714
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(7814..7885,1..308)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
       61 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      121 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      181 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      241 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      301 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      361 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      421 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
      481 gtgtacggtg ggaggtctat ataagcagag ctcaataaaa gagcccacaa cccctcactc
      541 ggcgcgccag tcttccgata gactgcgtcg cccgggtacc cgtattccca ataaagcctc
      601 ttgctgtttg catccgaatc gtggtctcgc tgttccttgg gagggtctcc tctgagtgat
      661 tgactaccca cgacgggggt ctttcatttg ggggctcgtc cgggatttgg agacccctgc
      721 ccagggacca ccgacccacc accgggaggt aagctggcca gcaacttatc tgtgtctgtc
      781 cgattgtcta gtgtctatgt ttgatgttat gcgcctgcgt ctgtactagt tagctaacta
      841 gctctgtatc tggcggaccc gtggtggaac tgacgagttc tgaacacccg gccgcaaccc
      901 tgggagacgt cccagggact ttgggggccg tttttgtggc ccgacctgag gaagggagtc
      961 gatgtggaat ccgaccccgt caggatatgt ggttctggta ggagacgaga acctaaaaca
     1021 gttcccgcct ccgtctgaat ttttgctttc ggtttggaac cgaagccgcg cgtcttgtct
     1081 gctgcagcgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc
     1141 tgaaaattag ggccagactg ttaccactcc cttaagtttg accttaggtc actggaaaga
     1201 tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt gggttacctt
     1261 ctgctctgca gaatggccaa cctttaacgt cggatggccg cgagacggca cctttaaccg
     1321 agacctcatc acccaggtta agatcaaggt cttttcacct ggcccgcatg gacacccaga
     1381 ccaggtcccc tacatcgtga cctgggaagc cttggctttt gacccccctc cctgggtcaa
     1441 gccctttgta caccctaagc ctccgcctcc tcttcctcca tccgccccgt ctctccccct
     1501 tgaacctcct cgttcgaccc cgcctcgatc ctccctttat ccagccctca ctccttctct
     1561 aggcgccgga attgaagatc tgggggatcg atcctctaga gtccgttaca taacttacgg
     1621 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     1681 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     1741 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     1801 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1861 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1921 ggcagtacat caatgggcgt gaatagcggt ttgactcacg gggatttcca agtctccacc
     1981 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     2041 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     2101 taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg
     2161 acctccatag aagacaccgg gaccgatcca gcctccgcgg ccgggaacgg tgcattggaa
     2221 cgctgcagga attgatccgc ggccgcaccg gtaggcctcg tacgcttaat taacggatcc
     2281 ggaattccgc ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata
     2341 aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt
     2401 gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct
     2461 cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc
     2521 ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga
     2581 caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc
     2641 ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt
     2701 attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg
     2761 gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaacgtct aggccccccg
     2821 aaccacgggg acgtggtttt cctttgaaaa acacgatgat aagcttgcca caaccatggc
     2881 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     2941 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3001 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     3061 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     3121 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     3181 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     3241 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     3301 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     3361 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     3421 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     3481 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     3541 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     3601 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     3661 gttcttctga gcgggatcgg ctagagtcga cccggacggg acgctcctgc gcctgataca
     3721 gaacgaattg cttgcaggca tctcatgagt gtgtcttccc gttttccgcc tgaggtcact
     3781 gcgtggatgg agcgctggcg cctgctgcgc gacggcgagc tgctcaccac ccactcgagg
     3841 gctgcagcgc tgcagaggcc gagtgcagaa ctgctccaaa gggacctcaa ggctttccga
     3901 gggacactag gctgactcca tcgagccagt gtagagataa gcttatcgat tagtccaatt
     3961 tgttaaagac aggatatcag tggtccaggc tctagttttg actcaacaat atcaccagct
     4021 gaagcctata gagtacgagc catagataaa ataaaagatt ttatttagtc tccagaaaaa
     4081 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gactcgagcg gccgccactg
     4141 tgctggatat caaccacttt gtacaagaaa gctgggtcta gatatctcga gtgcggccgc
     4201 gaattccacc acactggact agtggatccg agctcggtac caagcttttc caaaaagaag
     4261 cagcacgact tcttctctct tgaagaagaa gtcgtgctgc ttcgggatct ctatcactga
     4321 tagggaactt ataagattcc caaatccaaa gacatttcac gtttatggtg atttcccaga
     4381 acacatagcg acatgcaaat attgcagggc gccactcccc tgtccctcac agccatcttc
     4441 ctgccagggc gcacgcgcgc tgggtgttcc cgcctagtga cactgggccg cgattccttg
     4501 gagcgggttg atgacgtcag cgttcgaatt cggtaccgga tccagtcgac tgaattggtt
     4561 cctttaaagc ctgctttttt gtacaaactt gttgatatct gcagaattcc accacactgg
     4621 actagcttaa gtaacgccat tttgcaaggc atggaaaaat acataactga gaatagagaa
     4681 gttcagatca aggtcaggaa cagatggaac agggtcgacc ctagagaacc atcagatgtt
     4741 tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt
     4801 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc
     4861 cctcactcgg ggcgccagtc ctccgattga ctgagtcgcc cgggtacccg tgtatccaat
     4921 aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt tccttgggag ggtctcctct
     4981 gagtgattga ctacccgtca gcgggggtct ttcatttggg ggctcgtccg ggatcgggag
     5041 acccctgccc agggaccacc gacccaccac cgggaggtaa gctggctgcc tcgcgcgttt
     5101 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     5161 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     5221 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtagatcc ggctgtggaa
     5281 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     5341 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     5401 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     5461 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     5521 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     5581 aggctttttt ggaggcctag gcttttgcaa aaagcttact ggcttaacta tgcggcatca
     5641 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg
     5701 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5761 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5821 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5881 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5941 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6001 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6061 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6121 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6181 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6241 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6301 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     6361 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6421 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6481 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6541 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6601 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6661 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6721 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6781 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6841 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6901 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6961 aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7021 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7081 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7141 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7201 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7261 tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg
     7321 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7381 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7441 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7501 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7561 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7621 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     7681 acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attagcttgg
     7741 ccattgcata cgttgtatcc atatcataat atgtacattt atattggctc atgtccaaca
     7801 ttaccgccat gttgacattg attattgact agttattaat agtaatcaat tacggggtca
     7861 ttagttcata gcccatatat ggagt
//
