LOCUS       sequence-367432-        4748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid to endogenously knock out the YTHDF2 in Hek293 cells. 
            Puromycin and polyA sequence is inserted between two homology arms. 
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4748)
  AUTHORS   Meng Q, Stoyko D, Andrews CM, Konstantinidou P, Genzor P, O T, 
            Elchert AR, Benner L, Sobti S, Katz EY, Haase AD
  TITLE     Functional editing of endogenous genes through rapid selection of 
            cell pools (Rapid generation of endogenously tagged genes in 
            Drosophila ovarian somatic sheath cells).
  JOURNAL   Nucleic Acids Res. 2022 May 27. pii: 6594084. doi: 
            10.1093/nar/gkac448.
  PUBMED    35639929
REFERENCE   2  (bases 1 to 4748)
  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: "Nucleic 
            Acids Res. 2022 May 27. pii: 6594084. doi: 10.1093/nar/gkac448."
COMMENT     Sequence Label: Donor_YTHDF2_KO_Puro
FEATURES             Location/Qualifiers
     source          1..4748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     371..388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    505..526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(541..546,547..564,565..571)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..595
                     /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     584..606
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     603..619
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     603..619
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1324..1923
                     /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(1324..1343)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1820..1840
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1924..2058
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1961..1980)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2015..2034
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2751..2768)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2751..2767)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2760..2782)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2976..2995)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3095..3117
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3155..3173)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3241..3345
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3346..3414,3415..4206)
                     /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(3564..3583)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4377..4748,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      541 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      601 cacaggaaac agctatgacc atgattacgc catgagggac tgtgtgactc cctaggggtc
      661 gggttacctc ccagatgggg gtggggtatc acttgcagct ggaaggcagg ttcattttaa
      721 ggtctcgcgg gcttcggagg cccctccctc actgagaaac cggcttttgt cgttcttctg
      781 aagccagagc tagtctttcc aggtgttagt cgaaacctcg tggtgcgacc ctggtcgtcc
      841 caaaccccct aggccttaat cctggggcgg tgggggcggg gaggccgtga gcacggcttc
      901 cgctcctcca atccgccaga gggcgcagcg gccggcctct cccttcccgg ggttcttcgc
      961 gccgggcccc ttccgcgtgg gtgagtgaat gtgagagtca gcgctcgcgc cgcgcgcgcc
     1021 gcccgcctcc gctgttcggc gctctgcttt aggcggtggg gggcgggcgc gcgcgtaaaa
     1081 gcatagagac gggcattgag ctcttgggct agagcgtcgc cgagtcggag ccggagcctg
     1141 agccgcgcgc tgtgtctccg ctgcgtccgc cgaggccccc gagtgtcagg gacaaaagcc
     1201 tccgcctgct cccgcagccg gggctcatct gccgccgccg ccgcgctgag gagagttcgc
     1261 cgccgtcgcc gcccgtgagg atctgagagc caattagctt gaccaagctt gctagcaatc
     1321 aaaatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt cccccgggcc
     1381 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgacccg
     1441 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     1501 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     1561 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     1621 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     1681 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     1741 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     1801 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     1861 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     1921 tagaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     1981 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     2041 tcttatcatg tctggatcaa gcgccccggg cggaggacct ttcggaagcc gcttagttcg
     2101 caggtgccgc acacttaagt attttgaccc tttcggtgtg ttcttgcagc tggcgaacag
     2161 ctttttcgct aacaaggagt aattcattaa gggtgggggt ggattgggtt ttgaaacgct
     2221 ccaggtcctt agtttcctgt aggtcttaga aggcctttgt ttttcatcct cgtggatcac
     2281 tctctggaag tactctcatg tgatggggga ggggaagcgg gagggaatgg aaatccagag
     2341 gagccaataa aatgaccttt ttagatcaac ttgctctggg tggaggggga cattttcact
     2401 ggcgactgtc gcttcagcat tcactgtcat ctcttaccct tttgaagttc aatcttacgt
     2461 gctttgttaa ctagtaaggt ttttctctgg ttagcatata tgaactaatt tgaaaggaag
     2521 attgtaggat tgggacactc ctaattgaat ttttttttct ttagcaaaat ggatctgtac
     2581 atcaaaagga tggattaaac gatgatgatt ttgaacctta cttgagtcca caggcaaggc
     2641 ccgtgagtag ttaactattt acatatctaa tcgaagggtg tggtatattc tttctccgcc
     2701 ttctaccaat aaatcccact agaggatccc cgggtaccga gctcgaattc actggccgtc
     2761 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     2821 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     2881 cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg
     2941 tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3001 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3061 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3121 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     3181 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     3241 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3301 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     3361 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     3421 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3481 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3541 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     3601 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     3661 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3721 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3781 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3841 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     3901 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3961 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     4021 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     4081 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     4141 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     4201 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     4261 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4321 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4381 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4441 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4501 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4561 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4621 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4681 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4741 accgaact
//
