LOCUS       sequence-315709-        7085 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Suppress MUTYH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7085)
  AUTHORS   Sviderskiy VO, Blumenberg L, Gorodetsky E, Karakousi TR, Hirsh N, 
            Alvarez SW, Terzi EM, Kaparos E, Whiten GC, Ssebyala S, Tonzi P, Mir
            H, Neel BG, Huang TT, Adams S, Ruggles KV, Possemato R
  TITLE     Hyperactive CDK2 Activity in Basal-like Breast Cancer Imposes a 
            Genome Integrity Liability that Can Be Exploited by Targeting DNA 
            Polymerase epsilon.
  JOURNAL   Mol Cell. 2020 Oct 28. pii: S1097-2765(20)30723-1. doi: 
            10.1016/j.molcel.2020.10.016.
  PUBMED    33152268
REFERENCE   2  (bases 1 to 7085)
  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: "Mol Cell. 
            2020 Oct 28. pii: S1097-2765(20)30723-1. doi: 
            10.1016/j.molcel.2020.10.016."
COMMENT     Sequence Label: pLKO.1P MUTYH_2
FEATURES             Location/Qualifiers
     source          1..7085
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    347..464
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        513..1023
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     942..960
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1045..1644
                     /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(1045..1064)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1541..1561
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1772..2005
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2077..2198
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2114..2133)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2168..2187
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2238..2373
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2393..2412)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2394..2412)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2422..2439)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2422..2438)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2431..2453)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2580..3035
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2667..2686)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2877..2898
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3061..3165
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3166..3234,3235..4026)
                     /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(3384..3403)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4197..4785
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4686..4705
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4939..4956
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5073..5094
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5109..5114,5115..5132,5133..5139)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5147..5163
                     /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     5152..5174
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5171..5187
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5171..5187
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5206..5226
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5208..5226
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5254..5480
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5481..5661
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5708..5833
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6326..6559
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6744..6788
                     /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 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gccactgtga tcaactacta tactcgaagt atagtagttg atcacagtgg
      301 tttttgaatt ctcgacctcg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
      361 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
      421 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
      481 acagcagaga tccactttgg ccgcggctcg agggggttgg ggttgcgcct tttccaaggc
      541 agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa acgcagcggc
      601 gccgaccctg ggtctcgcac attcttcacg tccgttcgca gcgtcacccg gatcttcgcc
      661 gctacccttg tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc
      721 ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg cacgtctcac tagtaccctc
      781 gcagacggac agcgccaggg agcaatggca gcgcgccgac cgcgatgggc tgtggccaat
      841 agcggctgct cagcagggcg cgccgagagc agcggccggg aaggggcggt gcgggaggcg
      901 gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag
      961 cctccggagc gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga cctctctccc
     1021 cagggggatc caccggagct taccatgacc gagtacaagc ccacggtgcg cctcgccacc
     1081 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     1141 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     1201 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     1261 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     1321 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     1381 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     1441 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     1501 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     1561 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     1621 atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg
     1681 agcgcacgac cccatgcatc ggtaccttta agaccaatga cttacaaggc agctgtagat
     1741 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     1801 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     1861 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     1921 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1981 tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat tcagtattta
     2041 taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat
     2101 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     2161 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc
     2221 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     2281 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     2341 tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag
     2401 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     2461 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     2521 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     2581 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2641 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2701 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2761 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2821 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2881 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2941 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3001 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     3061 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3121 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     3181 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     3241 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3301 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3361 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     3421 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     3481 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3541 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3601 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3661 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     3721 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3781 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3841 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     3901 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3961 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     4021 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     4081 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4141 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4201 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4261 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4321 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4381 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4441 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4501 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4561 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4621 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4681 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4741 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4801 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4861 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4921 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     4981 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     5041 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     5101 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     5161 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
     5221 taaagggaac aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt
     5281 cttgcaacat ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg
     5341 catgccgatt ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg
     5401 gtctgacatg gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt
     5461 gcctagctcg atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct
     5521 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     5581 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     5641 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
     5701 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
     5761 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
     5821 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
     5881 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
     5941 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
     6001 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     6061 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     6121 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga
     6181 tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata
     6241 aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc
     6301 agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag
     6361 gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg
     6421 gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc
     6481 aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc
     6541 taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg
     6601 ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga
     6661 cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg
     6721 aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg
     6781 caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa
     6841 tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata
     6901 gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac
     6961 ccgacaggcc cgaaggaata gaagaagaag gtggagagag agacagagac agatccattc
     7021 gattagtgaa cggatctcga cggtatcgat cacgagacta gcctcgagcg gccgccccct
     7081 tcacc
//
