LOCUS       Exported                7080 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  PTEN shRNA.
ACCESSION   .
VERSION     .
KEYWORDS    pLKO.1 Puro shRNA PTEN KD4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7080)
  AUTHORS   Roman-Fernandez A, Roignot J, Sandilands E, Nacke M, Mansour MA, 
            McGarry L, Shanks E, Mostov KE, Bryant DM
  TITLE     The phospholipid PI(3,4)P2 is an apical identity determinant.
  JOURNAL   Nat Commun. 2018 Nov 28;9(1):5041. doi: 10.1038/s41467-018-07464-8.
  PUBMED    30487552
REFERENCE   2  (bases 1 to 7080)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7080
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    84..201
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        250..760
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     679..697
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             782..1381
                     /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(782..801)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1278..1298
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1509..1742
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1814..1935
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1851..1870)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1905..1924
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1975..2110
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2037..2056
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2130..2149)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2131..2149)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2159..2176)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2159..2175)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2168..2190)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2317..2772
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2404..2423)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2614..2635
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2798..2902
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2903..3763
                     /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(3121..3140)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3934..4522
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4423..4442
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4676..4693
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4810..4831
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4846..4876
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4884..4900
                     /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     4889..4911
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4908..4924
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4908..4924
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4943..4963
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4945..4963
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4991..5217
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5218..5398
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5445..5570
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6063..6296
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6481..6525
                     /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        6823..7063
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     6823..6843
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6994..7013
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 ggctagcagt tcctcgagga actgctagcc tccgggtttt ttgaattctc gacctcgaga
       61 caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca
      121 ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa
      181 attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggccg
      241 cggctcgagg gggttggggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg
      301 cggctgctct gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt
      361 cttcacgtcc gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc
      421 gacgcttcct gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac
      481 gtgacaaacg gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc
      541 aatggcagcg cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc
      601 cgagagcagc ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc
      661 cctgttcctg cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt
      721 cggctccctc gttgaccgaa tcaccgacct ctctccccag ggggatccac cggagcttac
      781 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
      841 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
      901 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
      961 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     1021 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     1081 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     1141 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     1201 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     1261 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     1321 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     1381 acgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcggt
     1441 acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa
     1501 ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac
     1561 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     1621 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     1681 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     1741 cagtagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc
     1801 agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     1861 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     1921 catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg
     1981 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     2041 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     2101 tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac
     2161 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     2221 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     2281 cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa
     2341 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2401 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2461 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2521 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2581 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2641 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     2701 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2761 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     2821 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     2881 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     2941 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3001 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3061 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     3121 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     3181 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     3241 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     3301 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3361 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     3421 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     3481 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     3541 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     3601 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     3661 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     3721 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     3781 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     3841 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     3901 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3961 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4021 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4081 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4141 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4201 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4261 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4321 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4381 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4441 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4501 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4561 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     4621 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     4681 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     4741 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     4801 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     4861 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     4921 tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct
     4981 gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt
     5041 tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg
     5101 tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc
     5161 actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg
     5221 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     5281 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     5341 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     5401 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
     5461 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
     5521 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
     5581 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     5641 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     5701 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     5761 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     5821 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     5881 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     5941 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     6001 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     6061 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     6121 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     6181 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     6241 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     6301 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     6361 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     6421 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     6481 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     6541 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     6601 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     6661 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     6721 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg
     6781 tatcgatcac gagactagcc tcgagcggcc gcccccttca ccgagggcct atttcccatg
     6841 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
     6901 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     6961 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     7021 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cggacccgga
//
