LOCUS       sequence-457049-        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_i2-ipUSEPR-TR657
FEATURES             Location/Qualifiers
     source          1..7842
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        618..816
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             834..1014
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1061..1186
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1683..1916
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2101..2145
                     /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    2443..2560
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2653..2901
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2653..2673
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2824..2843
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3069..3280
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             3331..3927
                     /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(3331..3350)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3827..3847
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3943..3999
                     /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             4006..4707
                     /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    4730..5318
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4783..4803)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5201..5212)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5321..5337)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5322..5338)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5847..6027
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6089..6677)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6169..6188)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6848..7639,7640..7708))
                     /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     7471..7490
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7709..7813)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc
      841 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      901 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      961 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     1021 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     1081 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     1141 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     1201 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     1261 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     1321 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     1381 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     1441 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     1501 aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat
     1561 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1621 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1681 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1741 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1801 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1861 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1921 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1981 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     2041 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     2101 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     2161 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     2221 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2281 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2341 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2401 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2461 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2521 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2581 cagagatcca gtttggttag taccgggccc gggtcgacta gtgatcagtg tgagggagtg
     2641 taaagctggt ttgagggcct atttcccatg attccttcat atttgcatat acgatacaag
     2701 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
     2761 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     2821 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     2881 ttgtggaaag gacgaaacac cggcgatatc cagacattca ggtttaagag ctatgctgga
     2941 aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     3001 tcggtgcttt tttaaaccgc tgttcctagg aatcccgagg ccttcgagtg gctccggtgc
     3061 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     3121 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     3181 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     3241 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac gcgggacccg
     3301 acattagcgc tacagcttaa gcgggccacc atgaccgagt acaagcccac ggtgcgcctc
     3361 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     3421 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     3481 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     3541 gccgccgtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     3601 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     3661 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc
     3721 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     3781 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     3841 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     3901 tggtgcatga cccgcaagcc cggtgccgct agcggcagcg gcgccaccaa cttcagcctg
     3961 ctgaagcagg ccggcgacgt ggaggagaac cccggccccg gatccatgag cgagctgatc
     4021 accgagaaca tgcacatgaa gctgtacatg gagggcaccg tgaacaacca ccacttcaag
     4081 tgcacatccg agggcgaagg caagccctac gagggcaccc agacccagag aatcaaggtg
     4141 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctaccagctt catgtacggc
     4201 agtcacacct tcatcaacca cacccagggc atccccgact tctggaagca gtccttccct
     4261 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
     4321 caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac
     4381 ttcccatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc ccacaccgag
     4441 atgctgtacc ccgctgacgg cggcctggaa ggcagaaccg cgctggccct gaagctcgtg
     4501 ggcgggggcc acctgatctg caacttcaag accacataca gatccaagaa acccgctaag
     4561 aacctcaaga tgcccggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc
     4621 gacaaagaga cctacgtcga gcagcacgag gtggctgtgg ccagatactg cgacctccct
     4681 agcaaactgg ggcacaagct taattaatct agaccgcggc gcgcccgata atcaacctct
     4741 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     4801 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     4861 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     4921 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     4981 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     5041 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     5101 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     5161 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     5221 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     5281 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgatc
     5341 gagacctaga aaaacatgga gcaatcacaa gtagcaatac agcagctacc aatgctgatt
     5401 gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca cctcaggtac
     5461 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     5521 ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat ctgtggatct
     5581 accacacaca aggctacttc cctgattggc agaactacac accagggcca gggatcagat
     5641 atccactgac ctttggatgg tgctacaagc tagtaccagt tgagcaagag aaggtagaag
     5701 aagccaatga aggagagaac acccgcttgt tacaccctgt gagcctgcat gggatggatg
     5761 acccggagag agaagtatta gagtggaggt ttgacagccg cctagcattt catcacatgg
     5821 cccgagagct gcatccggac tgtactgggt ctctctggtt agaccagatc tgagcctggg
     5881 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     5941 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     6001 tttagtcagt gtggaaaatc tctagcagca tgtgagcaaa aggccagcaa aaggccagga
     6061 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6121 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6181 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6241 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6301 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6361 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6421 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6481 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6541 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6601 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6661 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6721 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6781 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6841 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6901 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6961 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     7021 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7081 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7141 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7201 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7261 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7321 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7381 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7441 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7501 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7561 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7621 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7681 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7741 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7801 taggggttcc gcgcacattt ccccgaaaag tgccacctga cg
//
