LOCUS       Exported                7080 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  INPP5E shRNA.
ACCESSION   .
VERSION     .
KEYWORDS    pLKO.1 Puro shRNA INPP5E KD2
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    42..159
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        208..718
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     637..655
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             740..1339
                     /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(740..759)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1236..1256
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1467..1700
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1772..1893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1809..1828)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1863..1882
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1933..2068
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1995..2014
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2088..2107)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2089..2107)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2117..2134)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2117..2133)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2126..2148)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2275..2730
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2362..2381)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2572..2593
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2756..2860
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2861..3721
                     /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(3079..3098)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3892..4480
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4381..4400
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4634..4651
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4768..4789
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4804..4834
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4842..4858
                     /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     4847..4869
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4866..4882
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4866..4882
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4901..4921
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4903..4921
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4949..5175
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5176..5356
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5403..5528
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6021..6254
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6439..6483
                     /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        6781..7021
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     6781..6801
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6952..6971
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gaattctcga cctcgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg
       61 attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact
      121 aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc
      181 agagatccac tttggccgcg gctcgagggg gttggggttg cgccttttcc aaggcagccc
      241 tgggtttgcg cagggacgcg gctgctctgg gcgtggttcc gggaaacgca gcggcgccga
      301 ccctgggtct cgcacattct tcacgtccgt tcgcagcgtc acccggatct tcgccgctac
      361 ccttgtgggc cccccggcga cgcttcctgc tccgccccta agtcgggaag gttccttgcg
      421 gttcgcggcg tgccggacgt gacaaacgga agccgcacgt ctcactagta ccctcgcaga
      481 cggacagcgc cagggagcaa tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg
      541 ctgctcagca gggcgcgccg agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg
      601 tggggcggta gtgtgggccc tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc
      661 ggagcgcacg tcggcagtcg gctccctcgt tgaccgaatc accgacctct ctccccaggg
      721 ggatccaccg gagcttacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
      781 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
      841 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
      901 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
      961 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     1021 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     1081 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca
     1141 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     1201 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     1261 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     1321 ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc
     1381 acgaccccat gcatcggtac ctttaagacc aatgacttac aaggcagctg tagatcttag
     1441 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
     1501 tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     1561 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     1621 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     1681 agtgtggaaa atctctagca gtagtagttc atgtcatctt attattcagt atttataact
     1741 tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta
     1801 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     1861 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc
     1921 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     1981 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     2041 aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt
     2101 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     2161 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     2221 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc
     2281 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2341 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2401 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2461 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2521 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2581 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2641 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2701 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     2761 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     2821 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     2881 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     2941 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3001 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3061 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3121 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3181 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3241 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3301 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3361 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     3421 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3481 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3541 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     3601 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     3661 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     3721 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     3781 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     3841 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3901 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3961 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4021 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4081 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4141 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4201 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4261 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4321 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4381 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4441 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4501 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4561 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4621 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     4681 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     4741 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     4801 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     4861 tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag
     4921 ggaacaaaag ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc
     4981 aacatggtaa cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc
     5041 cgattggtgg aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg
     5101 acatggattg gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta
     5161 gctcgataca taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     5221 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     5281 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     5341 tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg
     5401 agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc
     5461 gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg
     5521 agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc
     5581 cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac
     5641 gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac
     5701 agctacaacc atcccttcag acaggatcag aagaacttag atcattatat aatacagtag
     5761 caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca
     5821 agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc
     5881 agacctggag gaggagatat gagggacaat tggagaagtg aattatataa atataaagta
     5941 gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga
     6001 gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc
     6061 actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata
     6121 gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc
     6181 acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag
     6241 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg
     6301 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg
     6361 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa
     6421 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt
     6481 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata
     6541 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt
     6601 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac
     6661 aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta
     6721 gtgaacggat ctcgacggta tcgatcacga gactagcctc gagcggccgc ccccttcacc
     6781 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
     6841 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
     6901 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
     6961 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
     7021 cgaaacaccg gagggtccat cttcaagggc ctcgaggccc ttgaagatgg accctttttt
//
