LOCUS       sequence-457225-        7842 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockdown of Cdkn1a in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7842)
  AUTHORS   Lin L, Zhao J, Kubota N, Li Z, Lam YL, Nguyen LP, Yang L, Pokharel 
            SP, Blue SM, Yee BA, Chen R, Yeo GW, Chen CW, Chen L, Zheng S
  TITLE     Epistatic interactions between NMD and TRP53 control progenitor cell
            maintenance and brain size.
  JOURNAL   Neuron. 2024 Jul 3;112(13):2157-2176.e12. doi: 
            10.1016/j.neuron.2024.04.006. Epub 2024 May 1.
  PUBMED    38697111
REFERENCE   2  (bases 1 to 7842)
  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"; doi: 
            "10.1016/j.neuron.2024.04.006"; journalName: "Neuron"; date: 
            "2024-07-3- 3"; volume: "112"; issue: "13"; pages: "2157-2176.e12"
COMMENT     Sequence Label: sg_Cdkn1a_i1-ipUSEPR-TR657
FEATURES             Location/Qualifiers
     source          1..7842
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /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"
     misc_feature    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2654..2902
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2654..2674
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2825..2844
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3070..3281
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             3332..3928
                     /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(3332..3351)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3828..3848
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3944..4000
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4007..4708
                     /codon_start=1
                     /product="far-red monomeric fluorescent protein with an 
                     emission peak at 657 nm (Morozova et al., 2010)"
                     /label=TagRFP657
                     /note="derived from mKate"
                     /translation="MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTQRIKV
                     VEGGPLPFAFDILATSFMYGSHTFINHTQGIPDFWKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEAHTEMLYPADGGLEGRTALALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDYRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    4731..5319
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4784..4804)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5202..5213)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5322..5338)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5323..5339)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5848..6028
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6090..6678)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6170..6189)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6849..7640,7641..7709))
                     /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     7472..7491
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7710..7814)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta gtaccgggcc cgggtcgact agtgatcagt gtgagggagt
     2641 gtaaagctgg tttgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     2701 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     2761 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     2821 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     2881 cttgtggaaa ggacgaaaca ccgccgcggt gtcagagtct aggtttaaga gctatgctgg
     2941 aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     3001 gtcggtgctt ttttaaaccg ctgttcctag gaatcccgag gccttcgagt ggctccggtg
     3061 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
     3121 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
     3181 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
     3241 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtgtcgtga cgcgggaccc
     3301 gacattagcg ctacagctta agcgggccac catgaccgag tacaagccca cggtgcgcct
     3361 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     3421 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     3481 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     3541 cgccgccgtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     3601 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     3661 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt
     3721 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     3781 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     3841 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     3901 ctggtgcatg acccgcaagc ccggtgccgc tagcggcagc ggcgccacca acttcagcct
     3961 gctgaagcag gccggcgacg tggaggagaa ccccggcccc ggatccatga gcgagctgat
     4021 caccgagaac atgcacatga agctgtacat ggagggcacc gtgaacaacc accacttcaa
     4081 gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagacccaga gaatcaaggt
     4141 ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctaccagct tcatgtacgg
     4201 cagtcacacc ttcatcaacc acacccaggg catccccgac ttctggaagc agtccttccc
     4261 tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac
     4321 ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa
     4381 cttcccatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg cccacaccga
     4441 gatgctgtac cccgctgacg gcggcctgga aggcagaacc gcgctggccc tgaagctcgt
     4501 gggcgggggc cacctgatct gcaacttcaa gaccacatac agatccaaga aacccgctaa
     4561 gaacctcaag atgcccggcg tctactatgt ggactacaga ctggaaagaa tcaaggaggc
     4621 cgacaaagag acctacgtcg agcagcacga ggtggctgtg gccagatact gcgacctccc
     4681 tagcaaactg gggcacaagc ttaattaatc tagaccgcgg cgcgcccgat aatcaacctc
     4741 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     4801 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     4861 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     4921 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     4981 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     5041 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     5101 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     5161 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     5221 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     5281 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgat
     5341 cgagacctag aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat
     5401 tgtgcctggc tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta
     5461 cctttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     5521 gggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga tctgtggatc
     5581 taccacacac aaggctactt ccctgattgg cagaactaca caccagggcc agggatcaga
     5641 tatccactga cctttggatg gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa
     5701 gaagccaatg aaggagagaa cacccgcttg ttacaccctg tgagcctgca tgggatggat
     5761 gacccggaga gagaagtatt agagtggagg tttgacagcc gcctagcatt tcatcacatg
     5821 gcccgagagc tgcatccgga ctgtactggg tctctctggt tagaccagat ctgagcctgg
     5881 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     5941 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     6001 ttttagtcag tgtggaaaat ctctagcagc atgtgagcaa aaggccagca aaaggccagg
     6061 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6121 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6181 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6241 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6301 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6361 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6421 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6481 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6541 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6601 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6661 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6721 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6781 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6841 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6901 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6961 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     7021 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     7081 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7141 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7201 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7261 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7321 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7381 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7441 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7501 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7561 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7621 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7681 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7741 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7801 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ac
//
