LOCUS       sequence-318825-        7080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PTEN shRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 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 PTEN KD2
FEATURES             Location/Qualifiers
     source          1..7080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    123..144
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(159..164,165..182,183..189)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    197..213
                     /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     202..224
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     221..237
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     221..237
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     256..276
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        258..276
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        304..530
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             531..711
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    758..883
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1376..1609
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1794..1838
                     /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        2136..2384
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2136..2156
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2307..2326
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2477..2594
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2643..3153
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3072..3090
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3175..3774
                     /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(3175..3194)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3671..3691
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3902..4135
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4207..4328
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4244..4263)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4298..4317
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4368..4503
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4523..4542)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4524..4542)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4552..4569)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4552..4568)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4561..4583)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4710..5165
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4797..4816)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5007..5028
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5191..5295
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5296..5364,5365..6156)
                     /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(5514..5533)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6327..6915
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6816..6835
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7069..7080,1..6)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
       61 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
      121 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
      181 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
      241 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct
      301 taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac
      361 atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg
      421 atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga accactgaat
      481 tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct
      541 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
      601 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
      661 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
      721 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
      781 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
      841 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
      901 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
      961 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
     1021 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
     1081 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
     1141 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
     1201 taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg
     1261 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1321 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1381 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     1441 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     1501 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     1561 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     1621 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     1681 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     1741 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     1801 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     1861 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     1921 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     1981 ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag
     2041 gtggagagag agacagagac agatccattc gattagtgaa cggatctcga cggtatcgat
     2101 cacgagacta gcctcgagcg gccgccccct tcaccgaggg cctatttccc atgattcctt
     2161 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     2221 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     2281 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     2341 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggtcca ccacagctag
     2401 aacttctcga gaagttctag ctgtggagga tttttgaatt ctcgacctcg agacaaatgg
     2461 cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa
     2521 gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa
     2581 aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg ccgcggctcg
     2641 agggggttgg ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc
     2701 tctgggcgtg gttccgggaa acgcagcggc gccgaccctg ggtctcgcac attcttcacg
     2761 tccgttcgca gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt
     2821 cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa
     2881 acggaagccg cacgtctcac tagtaccctc gcagacggac agcgccaggg agcaatggca
     2941 gcgcgccgac cgcgatgggc tgtggccaat agcggctgct cagcagggcg cgccgagagc
     3001 agcggccggg aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc
     3061 ctgcccgcgc ggtgttccgc attctgcaag cctccggagc gcacgtcggc agtcggctcc
     3121 ctcgttgacc gaatcaccga cctctctccc cagggggatc caccggagct taccatgacc
     3181 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     3241 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     3301 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     3361 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     3421 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     3481 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     3541 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     3601 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     3661 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     3721 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg
     3781 ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc ggtaccttta
     3841 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     3901 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct
     3961 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     4021 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     4081 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt
     4141 agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt
     4201 gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4261 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4321 gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa
     4381 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     4441 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     4501 gcctagggac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt
     4561 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     4621 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     4681 acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     4741 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     4801 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4861 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4921 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4981 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5041 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     5101 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     5161 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5221 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5281 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5341 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5401 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5461 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5521 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5581 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5641 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5701 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5761 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5821 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5881 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5941 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6001 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6061 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6121 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6181 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6241 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6301 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6361 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6421 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6481 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6541 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6601 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6661 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6721 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6781 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6841 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6901 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6961 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     7021 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
//
