LOCUS       sequence-322380-        7084 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Suppress POLE1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7084)
  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 7084)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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 POLE1_2
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             41..85
                     /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        383..631
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     383..403
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     554..573
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    728..845
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        894..1404
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1323..1341
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1426..2025
                     /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(1426..1445)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1922..1942
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2153..2386
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2458..2579
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2495..2514)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2549..2568
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2619..2754
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2774..2793)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2775..2793)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2803..2820)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2803..2819)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2812..2834)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2961..3416
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3048..3067)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3258..3279
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3442..3546
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3547..3615,3616..4407)
                     /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(3765..3784)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4578..5166
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5067..5086
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5320..5337
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5454..5475
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5490..5495,5496..5513,5514..5520)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5528..5544
                     /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     5533..5555
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5552..5568
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5552..5568
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5587..5607
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5589..5607
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5635..5861
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5862..6042
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6089..6214
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6707..6940
                     /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 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
       61 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
      121 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
      181 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
      241 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
      301 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac gagactagcc
      361 tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat atttgcatat
      421 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
      481 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      541 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      601 tttatatatc ttgtggaaag gacgaaacac cgggctgacc agttggacaa cattctcgag
      661 aatgttgtcc aactggtcag ctttttgaat tctcgacctc gagacaaatg gcagtattca
      721 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
      781 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
      841 attttcgggt ttattacagg gacagcagag atccactttg gccgcggctc gagggggttg
      901 gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt
      961 ggttccggga aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc
     1021 agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg
     1081 cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc
     1141 gcacgtctca ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga
     1201 ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg
     1261 gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg
     1321 cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac
     1381 cgaatcaccg acctctctcc ccagggggat ccaccggagc ttaccatgac cgagtacaag
     1441 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     1501 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     1561 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     1621 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     1681 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     1741 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     1801 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     1861 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     1921 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     1981 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacgccc gccccacgac
     2041 ccgcagcgcc cgaccgaaag gagcgcacga ccccatgcat cggtaccttt aagaccaatg
     2101 acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg
     2161 ctaattcact cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta
     2221 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     2281 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     2341 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtag tagttcatgt
     2401 catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac
     2461 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     2521 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     2581 catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca
     2641 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     2701 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga
     2761 cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca
     2821 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     2881 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     2941 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3001 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3061 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3121 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3181 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3241 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3301 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3361 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     3421 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3481 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3541 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     3601 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     3661 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3721 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3781 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3841 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3901 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     3961 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4021 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4081 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4141 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4201 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4261 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     4321 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     4381 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     4441 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4501 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4561 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4621 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4681 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4741 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4801 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4861 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4921 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4981 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5041 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5101 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5161 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5221 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     5281 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     5341 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     5401 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     5461 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     5521 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     5581 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt
     5641 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac
     5701 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct
     5761 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt
     5821 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc
     5881 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     5941 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     6001 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     6061 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     6121 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     6181 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     6241 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     6301 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     6361 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     6421 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     6481 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     6541 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     6601 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     6661 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     6721 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     6781 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     6841 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     6901 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     6961 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     7021 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     7081 gctt
//
