LOCUS       sequence-315539-        7322 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Suppress POLE1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7322)
  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 7322)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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.1B POLE1_1
FEATURES             Location/Qualifiers
     source          1..7322
                     /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    346..463
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        512..1022
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     941..959
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1034..1429
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    1463..2051
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1516..1536)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1934..1945)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2123..2356
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2434..2555
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2471..2490)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2525..2544
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2595..2730
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2750..2769)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2751..2769)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2779..2796)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2779..2795)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2788..2810)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2937..3392
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3024..3043)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3234..3255
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3418..3522
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3523..3591,3592..4383)
                     /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(3741..3760)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4554..5142
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5043..5062
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5296..5313
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5430..5451
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5466..5471,5472..5489,5490..5496)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5504..5520
                     /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     5509..5531
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5528..5544
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5528..5544
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5563..5583
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5565..5583
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5611..5837
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5838..6018
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6065..6190
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6683..6916
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7101..7145
                     /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 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaggtaccg ggcagtggat tactacttta ttctcgagaa taaagtagta atccactgct
      301 ttttgaattc tagatcttga gacaaatggc agtattcatc cacaatttta aaagaaaagg
      361 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
      421 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
      481 cagcagagat ccactttggc gccggctcga gggggcccgg gttgcgcctt ttccaaggca
      541 gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg
      601 ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg
      661 ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct
      721 tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact agtaccctcg
      781 cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata
      841 gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg
      901 ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc
      961 ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc
     1021 agggggatcc accatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc
     1081 aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct
     1141 ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg
     1201 tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat
     1261 cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt
     1321 gcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac
     1381 ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggcc tgcagctgca
     1441 gtagtaaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac
     1501 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     1561 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     1621 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     1681 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     1741 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     1801 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     1861 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     1921 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     1981 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     2041 cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac
     2101 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg
     2161 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     2221 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     2281 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     2341 tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa
     2401 cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt
     2461 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     2521 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc
     2581 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     2641 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     2701 agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga
     2761 gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     2821 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     2881 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc
     2941 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3001 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3061 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3121 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3181 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3241 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3301 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3361 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     3421 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3481 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     3541 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     3601 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3661 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3721 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     3781 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3841 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3901 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     3961 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4021 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     4081 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4141 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4201 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     4261 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4321 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4381 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4441 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4501 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4561 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4621 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4681 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     4741 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4801 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4861 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4921 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4981 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5041 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5101 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5161 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     5221 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     5281 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     5341 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     5401 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     5461 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     5521 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
     5581 agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt
     5641 gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat
     5701 gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc
     5761 tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc
     5821 tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     5881 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     5941 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     6001 tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga
     6061 ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg
     6121 gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg
     6181 cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag
     6241 gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga
     6301 acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg
     6361 acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt
     6421 agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga
     6481 caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct
     6541 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag
     6601 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
     6661 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
     6721 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta
     6781 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
     6841 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa
     6901 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
     6961 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct
     7021 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
     7081 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
     7141 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga
     7201 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag
     7261 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggaccca
     7321 ga
//
