LOCUS       sequence-320558-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PIK3C2gamma 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 PIK3C2G KD1
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        231..479
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     231..251
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     402..421
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    576..693
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        742..1252
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1171..1189
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1274..1873
                     /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(1274..1293)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1770..1790
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2001..2234
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2306..2427
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2343..2362)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2397..2416
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2467..2602
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2622..2641)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2623..2641)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2651..2668)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2651..2667)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2660..2682)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2809..3264
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2896..2915)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3106..3127
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3290..3394
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3395..3463,3464..4255)
                     /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(3613..3632)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4426..5014
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4915..4934
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5168..5185
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5302..5323
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5338..5343,5344..5361,5362..5368)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5376..5392
                     /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     5381..5403
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5400..5416
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5400..5416
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5435..5455
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5437..5455
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5483..5709
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5710..5890
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5937..6062
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6555..6788
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6973..7017
                     /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"
ORIGIN
        1 tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat
       61 attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag
      121 gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat
      181 ctcgacggta tcgatcacga gactagcctc gagcggccgc ccccttcacc gagggcctat
      241 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
      301 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      361 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      421 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
      481 gcctggaagc aacaagtcat ttctcgagaa atgacttgtt gcttccaggt ttttgaattc
      541 tcgacctcga gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
      601 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
      661 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
      721 ccactttggc cgcggctcga gggggttggg gttgcgcctt ttccaaggca gccctgggtt
      781 tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg
      841 gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt
      901 gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc
      961 ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact agtaccctcg cagacggaca
     1021 gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata gcggctgctc
     1081 agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg ggtgtggggc
     1141 ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg
     1201 cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc agggggatcc
     1261 accggagctt accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     1321 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     1381 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     1441 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     1501 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     1561 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     1621 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg
     1681 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     1741 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     1801 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     1861 gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc
     1921 ccatgcatcg gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     1981 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct
     2041 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     2101 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     2161 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     2221 gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa
     2281 gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata
     2341 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     2401 tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact
     2461 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     2521 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     2581 tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt
     2641 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     2701 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     2761 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
     2821 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2881 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2941 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3001 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3061 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3121 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3181 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     3241 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     3301 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     3361 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     3421 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     3481 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     3541 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     3601 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     3661 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     3721 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     3781 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     3841 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     3901 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     3961 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     4021 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     4081 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     4141 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     4201 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     4261 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     4321 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     4381 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4441 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4501 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4561 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4621 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4681 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4741 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4801 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4861 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4921 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4981 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5041 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5101 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     5161 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     5221 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     5281 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     5341 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     5401 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
     5461 aaagctggag ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg
     5521 gtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc atgccgattg
     5581 gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacggg tctgacatgg
     5641 attggacgaa ccactgaatt gccgcattgc agagatattg tatttaagtg cctagctcga
     5701 tacataaacg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     5761 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     5821 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     5881 atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct
     5941 ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
     6001 tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg
     6061 tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg
     6121 gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg
     6181 cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac
     6241 aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc
     6301 tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag
     6361 aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct
     6421 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     6481 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     6541 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     6601 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     6661 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     6721 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     6781 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     6841 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     6901 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     6961 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     7021 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     7081 ggct
//
