LOCUS       Exported                7081 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  3rd gen lentiviral vector for knocking down p53 gene expression.
ACCESSION   .
VERSION     .
KEYWORDS    shp53 pLKO.1 puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7081)
  AUTHORS   Godar S, Ince TA, Bell GW, Feldser D, Donaher JL, Bergh J, Liu A, 
            Miu K, Watnick RS, Reinhardt F, McAllister SS, Jacks T, Weinberg RA
  TITLE     Growth-inhibitory and tumor- suppressive functions of p53 depend on 
            its repression of CD44 expression.
  JOURNAL   Cell. 2008 Jul 11. 134(1):62-73.
  PUBMED    18614011
REFERENCE   2  (bases 1 to 7081)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7081
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     240..260
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        288..514
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             515..695
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    742..867
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1360..1593
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1778..1822
                     /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"
     promoter        2120..2360
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2120..2140
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2291..2310
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2462..2579
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2628..3138
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3057..3075
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3160..3759
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3160..3179)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3656..3676
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3887..4120
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4192..4313
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4229..4248)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4283..4302
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4353..4488
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4415..4434
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(4508..4527)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4509..4527)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4537..4554)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4537..4553)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4546..4568)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4695..5150
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4782..4801)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4992..5013
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5176..5280
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5281..6141
                     /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(5499..5518)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6312..6900
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6801..6820
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7054..7071
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
      241 caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc
      301 aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga
      361 gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg
      421 aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga
      481 tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg
      541 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
      601 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
      661 cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa
      721 gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg
      781 gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag
      841 aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg
      901 gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg
      961 gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc
     1021 tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga
     1081 tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac
     1141 accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag
     1201 caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga
     1261 attatataaa tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa
     1321 gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt
     1381 cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag
     1441 acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca
     1501 acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc
     1561 tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact
     1621 catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat
     1681 ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat
     1741 acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga
     1801 attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat
     1861 aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact
     1921 ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc
     1981 aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag
     2041 agacagatcc attcgattag tgaacggatc tcgacggtat cgatcacgag actagcctcg
     2101 agcggccgcc cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     2161 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     2221 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     2281 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     2341 atatatcttg tggaaaggac gaaacaccga ctccagtggt aatctacttc aagagagtag
     2401 attaccactg gagtcttttt gaattctcga cctcgagaca aatggcagta ttcatccaca
     2461 attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
     2521 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc
     2581 gggtttatta cagggacagc agagatccac tttggccgcg gctcgagggg gttggggttg
     2641 cgccttttcc aaggcagccc tgggtttgcg cagggacgcg gctgctctgg gcgtggttcc
     2701 gggaaacgca gcggcgccga ccctgggtct cgcacattct tcacgtccgt tcgcagcgtc
     2761 acccggatct tcgccgctac ccttgtgggc cccccggcga cgcttcctgc tccgccccta
     2821 agtcgggaag gttccttgcg gttcgcggcg tgccggacgt gacaaacgga agccgcacgt
     2881 ctcactagta ccctcgcaga cggacagcgc cagggagcaa tggcagcgcg ccgaccgcga
     2941 tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg agagcagcgg ccgggaaggg
     3001 gcggtgcggg aggcggggtg tggggcggta gtgtgggccc tgttcctgcc cgcgcggtgt
     3061 tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg gctccctcgt tgaccgaatc
     3121 accgacctct ctccccaggg ggatccaccg gagcttacca tgaccgagta caagcccacg
     3181 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     3241 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     3301 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     3361 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     3421 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     3481 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     3541 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     3601 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     3661 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     3721 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag
     3781 cgcccgaccg aaaggagcgc acgaccccat gcatcggtac ctttaagacc aatgacttac
     3841 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     3901 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
     3961 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     4021 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     4081 tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt
     4141 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt
     4201 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4261 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4321 tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4381 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4441 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc
     4501 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg
     4561 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     4621 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     4681 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4741 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4801 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4861 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     4921 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4981 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     5041 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5101 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     5161 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5221 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5281 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5341 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5401 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5461 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5521 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5581 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5641 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5701 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5761 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5821 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5881 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5941 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6001 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6061 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6121 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6181 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6241 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6301 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6361 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6421 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6481 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6541 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6601 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6661 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6721 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6781 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6841 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6901 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6961 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     7021 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     7081 g
//
