LOCUS       Exported                7080 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  INPP5E shRNA.
ACCESSION   .
VERSION     .
KEYWORDS    pLKO.1 Puro shRNA INPP5E KD1
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    47..164
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        213..723
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     642..660
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             745..1344
                     /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(745..764)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1241..1261
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1472..1705
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1777..1898
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1814..1833)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1868..1887
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1938..2073
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2000..2019
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2093..2112)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2094..2112)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2122..2139)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2122..2138)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2131..2153)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2280..2735
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2367..2386)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2577..2598
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2761..2865
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2866..3726
                     /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(3084..3103)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3897..4485
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4386..4405
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4639..4656
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4773..4794
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4809..4839
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4847..4863
                     /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     4852..4874
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4871..4887
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4871..4887
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4906..4926
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4908..4926
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4954..5180
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5181..5361
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5408..5533
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6026..6259
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6444..6488
                     /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        6786..7026
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     6786..6806
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6957..6976
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 tttttgaatt ctcgacctcg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
       61 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
      121 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
      181 acagcagaga tccactttgg ccgcggctcg agggggttgg ggttgcgcct tttccaaggc
      241 agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa acgcagcggc
      301 gccgaccctg ggtctcgcac attcttcacg tccgttcgca gcgtcacccg gatcttcgcc
      361 gctacccttg tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc
      421 ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg cacgtctcac tagtaccctc
      481 gcagacggac agcgccaggg agcaatggca gcgcgccgac cgcgatgggc tgtggccaat
      541 agcggctgct cagcagggcg cgccgagagc agcggccggg aaggggcggt gcgggaggcg
      601 gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag
      661 cctccggagc gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga cctctctccc
      721 cagggggatc caccggagct taccatgacc gagtacaagc ccacggtgcg cctcgccacc
      781 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
      841 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
      901 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
      961 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     1021 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     1081 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     1141 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     1201 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     1261 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     1321 atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg
     1381 agcgcacgac cccatgcatc ggtaccttta agaccaatga cttacaaggc agctgtagat
     1441 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     1501 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     1561 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     1621 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1681 tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat tcagtattta
     1741 taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat
     1801 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     1861 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc
     1921 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     1981 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     2041 tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag
     2101 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     2161 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     2221 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     2281 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2341 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2401 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2461 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2521 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2581 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2641 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2701 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     2761 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     2821 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     2881 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     2941 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3001 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3061 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     3121 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     3181 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3241 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3301 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3361 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     3421 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3481 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3541 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     3601 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3661 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     3721 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     3781 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     3841 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3901 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3961 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4021 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4081 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4141 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4201 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4261 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4321 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4381 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4441 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4501 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4561 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4621 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     4681 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     4741 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     4801 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     4861 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
     4921 taaagggaac aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt
     4981 cttgcaacat ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg
     5041 catgccgatt ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg
     5101 gtctgacatg gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt
     5161 gcctagctcg atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct
     5221 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     5281 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     5341 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
     5401 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
     5461 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
     5521 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
     5581 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
     5641 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
     5701 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     5761 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     5821 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga
     5881 tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata
     5941 aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc
     6001 agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag
     6061 gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg
     6121 gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc
     6181 aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc
     6241 taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg
     6301 ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga
     6361 cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg
     6421 aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg
     6481 caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa
     6541 tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata
     6601 gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac
     6661 ccgacaggcc cgaaggaata gaagaagaag gtggagagag agacagagac agatccattc
     6721 gattagtgaa cggatctcga cggtatcgat cacgagacta gcctcgagcg gccgccccct
     6781 tcaccgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     6841 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     6901 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     6961 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     7021 aaggacgaaa caccggggtg agcgagaggc tgctgctcga gcagcagcct ctcgctcacc
//
