LOCUS       sequence-315728-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Suppress POLA1.
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 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 POLA1_1
FEATURES             Location/Qualifiers
     source          1..7084
                     /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             1044..1643
                     /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(1044..1063)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1540..1560
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1771..2004
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2076..2197
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2113..2132)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2167..2186
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2237..2372
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2392..2411)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2393..2411)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2421..2438)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2421..2437)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2430..2452)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2579..3034
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2666..2685)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2876..2897
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3060..3164
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3165..3233,3234..4025)
                     /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(3383..3402)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4196..4784
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4685..4704
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4938..4955
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5072..5093
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5108..5113,5114..5131,5132..5138)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5146..5162
                     /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     5151..5173
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5170..5186
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5170..5186
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5205..5225
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5207..5225
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5253..5479
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5480..5660
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5707..5832
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6325..6558
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6743..6787
                     /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 ggctggtgag aagtataaat atctcgagat atttatactt ctcaccagct
      301 ttttgaattc tcgacctcga gacaaatggc agtattcatc cacaatttta aaagaaaagg
      361 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
      421 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
      481 cagcagagat ccactttggc cgcggctcga gggggttggg 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 accggagctt accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     1081 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     1141 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     1201 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     1261 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     1321 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     1381 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
     1441 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     1501 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     1561 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     1621 tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga
     1681 gcgcacgacc ccatgcatcg gtacctttaa gaccaatgac ttacaaggca gctgtagatc
     1741 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     1801 aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc
     1861 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     1921 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     1981 agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat
     2041 aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg
     2101 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     2161 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc
     2221 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     2281 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     2341 ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt
     2401 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     2461 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     2521 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     2581 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     2641 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2701 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     2761 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     2821 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     2881 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     2941 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     3001 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     3061 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3121 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3181 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3241 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3301 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3361 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     3421 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3481 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3541 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3601 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3661 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3721 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3781 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3841 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3901 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3961 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4021 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     4081 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4141 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4201 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4261 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4321 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4381 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4441 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4501 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4561 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4621 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4681 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4741 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4801 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4861 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4921 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     4981 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5041 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     5101 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     5161 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
     5221 aaagggaaca aaagctggag ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc
     5281 ttgcaacatg gtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc
     5341 atgccgattg gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacggg
     5401 tctgacatgg attggacgaa ccactgaatt gccgcattgc agagatattg tatttaagtg
     5461 cctagctcga tacataaacg ggtctctctg gttagaccag atctgagcct gggagctctc
     5521 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     5581 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     5641 agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca
     5701 gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg
     5761 cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg
     5821 tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta
     5881 aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta
     5941 gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg
     6001 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
     6061 gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta
     6121 gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat
     6181 cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa
     6241 agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca
     6301 gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg
     6361 aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg
     6421 tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca
     6481 actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct
     6541 aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc
     6601 tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac
     6661 ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga
     6721 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
     6781 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
     6841 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
     6901 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc
     6961 cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg
     7021 attagtgaac ggatctcgac ggtatcgatc acgagactag cctcgagcgg ccgccccctt
     7081 cacc
//
