LOCUS       sequence-8645-ex        7985 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd gen lentiviral 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 7985)
  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 7985)
  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 Neo/pTER shEGFP#2 (w22-2)
FEATURES             Location/Qualifiers
     source          1..7985
                     /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        4814..5313
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4849..4872)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5246..5265
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             5327..6130
                     /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(5390..5409)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6000..6019
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    6264..6288
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    6420..6438
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(6484..6503)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    complement(6680..6704)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     LTR             6779..6959
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    7031..7152
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7068..7087)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7122..7141
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7171..7306
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7338..7357)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7339..7357)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7367..7384)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7367..7383)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7376..7398)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7528..7983
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7615..7634)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7825..7846
                     /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 tgcagatatc cggactagtg
     4801 atctaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
     4861 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
     4921 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
     4981 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
     5041 atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag
     5101 cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag
     5161 aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
     5221 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
     5281 ctgttctcct cttcctcatc tccgggcctt tcgacctgca gccaatatgg gatcggccat
     5341 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     5401 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     5461 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     5521 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     5581 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     5641 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     5701 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     5761 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     5821 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     5881 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     5941 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     6001 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     6061 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     6121 gttcttctga ggggatccgt cgagggcccg gtacctttaa gaccaatgac ttacaaggca
     6181 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct agactcgagc
     6241 ggccgccact gtgctggata tcaaccactt tgtacaagaa agctgggtct agatatctcg
     6301 agtgcggccg cgaattccac cacactggac tagtggatcc gagctcggta ccaagctttt
     6361 ccaaaaagct ggagtacaac tacaactctc ttgaagttgt agttgtactc cagcggggat
     6421 ctctatcact gatagggaac ttataagatt cccaaatcca aagacatttc acgtttatgg
     6481 tgatttccca gaacacatag cgacatgcaa atattgcagg gcgccactcc cctgtccctc
     6541 acagccatct tcctgccagg gcgcacgcgc gctgggtgtt cccgcctagt gacactgggc
     6601 cgcgattcct tggagcgggt tgatgacgtc agcgttcgaa ttcggtaccg gatccagtcg
     6661 actgaattgg ttcctttaaa gcctgctttt ttgtacaaac ttgttgatat ctgcagaatt
     6721 ccaccacact ggactagctc actcccaacg aagacaagat ctgctttttg cttgtactgg
     6781 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctgcctag ggaacccact
     6841 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     6901 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     6961 tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga
     7021 gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     7081 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     7141 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc cagttccgcc
     7201 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     7261 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcgtc
     7321 gagacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     7381 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     7441 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     7501 tgcgcagcct gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta agcgcggcgg
     7561 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     7621 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     7681 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     7741 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     7801 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     7861 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     7921 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     7981 tttcc
//
