LOCUS       sequence-318869-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PIK3C2alpha shRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7084)
  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 7084)
  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: "Nat 
            Commun."; date: "2018-11-28"; volume: "9(1):5041. doi"; pages: " 
            10.1038/s41467-018-07464-8"
COMMENT     Sequence Label: pLKO.1 Puro shRNA PIK3C2A KD5
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             61..105
                     /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        403..651
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     403..423
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     574..593
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    748..865
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        914..1424
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1343..1361
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1446..2045
                     /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(1446..1465)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1942..1962
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2173..2406
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2478..2599
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2515..2534)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2569..2588
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2639..2774
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2794..2813)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2795..2813)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2823..2840)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2823..2839)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2832..2854)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2981..3436
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3068..3087)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3278..3299
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3462..3566
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3567..3635,3636..4427)
                     /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(3785..3804)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4598..5186
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5087..5106
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5340..5357
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5474..5495
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5510..5515,5516..5533,5534..5540)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5548..5564
                     /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     5553..5575
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5572..5588
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5572..5588
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5607..5627
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5609..5627
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5655..5881
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5882..6062
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6109..6234
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6727..6960
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
ORIGIN
        1 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
       61 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
      121 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
      181 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
      241 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
      301 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg
      361 tatcgatcac gagactagcc tcgagcggcc gcccccttca ccgagggcct atttcccatg
      421 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
      481 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
      541 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
      601 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgggctcaga
      661 tatccagcaa taatctcgag attattgctg gatatctgag ctttttgaat tctcgacctc
      721 gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
      781 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
      841 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg
      901 gccgcggctc gagggggttg gggttgcgcc ttttccaagg cagccctggg tttgcgcagg
      961 gacgcggctg ctctgggcgt ggttccggga aacgcagcgg cgccgaccct gggtctcgca
     1021 cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc
     1081 cggcgacgct tcctgctccg cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc
     1141 ggacgtgaca aacggaagcc gcacgtctca ctagtaccct cgcagacgga cagcgccagg
     1201 gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc
     1261 gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt
     1321 gggccctgtt cctgcccgcg cggtgttccg cattctgcaa gcctccggag cgcacgtcgg
     1381 cagtcggctc cctcgttgac cgaatcaccg acctctctcc ccagggggat ccaccggagc
     1441 ttaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
     1501 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc
     1561 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     1621 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     1681 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     1741 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     1801 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc
     1861 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     1921 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     1981 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     2041 cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga ccccatgcat
     2101 cggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     2161 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
     2221 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     2281 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     2341 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     2401 ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
     2461 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     2521 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     2581 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
     2641 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     2701 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     2761 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
     2821 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     2881 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     2941 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     3001 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     3061 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3121 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3181 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3241 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3301 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     3361 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     3421 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3481 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3541 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3601 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3661 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3721 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3781 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     3841 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3901 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3961 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     4021 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     4081 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     4141 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     4201 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     4261 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     4321 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     4381 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4441 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4501 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4561 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4621 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4681 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4741 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4801 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4861 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4921 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4981 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     5041 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     5101 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     5161 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5221 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     5281 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     5341 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     5401 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     5461 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     5521 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     5581 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
     5641 agctgcaagc ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat
     5701 gagttagcaa catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt
     5761 aaggtggtac gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg
     5821 aaccactgaa ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa
     5881 cgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     5941 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     6001 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     6061 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
     6121 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
     6181 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
     6241 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
     6301 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
     6361 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
     6421 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     6481 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     6541 caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg
     6601 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     6661 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     6721 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     6781 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     6841 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     6901 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     6961 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     7021 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     7081 agaa
//
