LOCUS       sequence-367848-        5174 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid to endogenously knock out the YTHDF2 in Hek293 cells. 
            Hygromycin 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 5174)
  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 5174)
  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_Hygro
FEATURES             Location/Qualifiers
     source          1..5174
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(16..120)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     188..206
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(244..266)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     366..385
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     579..601
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     593..610
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     594..610
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1303..1437
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1327..1346)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1381..1400
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1438..2463)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(1438..2463)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(3168..3184)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3168..3184)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3181..3203)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3192..3208
                     /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)."
     promoter        complement(join(3216..3222,3223..3240,3241..3246))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3261..3282
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3399..3416)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3570..4158)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3650..3669)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4329..5120,5121..5174,1..15))
                     /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     4952..4971
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
       61 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
      121 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
      181 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
      241 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
      301 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
      361 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
      421 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
      481 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
      541 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
      601 gacggccagt gaattcgagc tcggtacccg gggatcctct agtgggattt attggtagaa
      661 ggcggagaaa gaatatacca cacccttcga ttagatatgt aaatagttaa ctactcacgg
      721 gccttgcctg tggactcaag taaggttcaa aatcatcatc gtttaatcca tccttttgat
      781 gtacagatcc attttgctaa agaaaaaaaa attcaattag gagtgtccca atcctacaat
      841 cttcctttca aattagttca tatatgctaa ccagagaaaa accttactag ttaacaaagc
      901 acgtaagatt gaacttcaaa agggtaagag atgacagtga atgctgaagc gacagtcgcc
      961 agtgaaaatg tccccctcca cccagagcaa gttgatctaa aaaggtcatt ttattggctc
     1021 ctctggattt ccattccctc ccgcttcccc tcccccatca catgagagta cttccagaga
     1081 gtgatccacg aggatgaaaa acaaaggcct tctaagacct acaggaaact aaggacctgg
     1141 agcgtttcaa aacccaatcc acccccaccc ttaatgaatt actccttgtt agcgaaaaag
     1201 ctgttcgcca gctgcaagaa cacaccgaaa gggtcaaaat acttaagtgt gcggcacctg
     1261 cgaactaagc ggcttccgaa aggtcctccg cccggggcgc ttgatccaga catgataaga
     1321 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     1381 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttcta
     1441 ttcctttgcc ctcggacgag tgctggggcg tcggtttcca ctatcggcga gtacttctac
     1501 acagccatcg gtccagacgg ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc
     1561 ggctccggat cggacgattg cgtcgcatcg accctgcgcc caagctgcat catcgaaatt
     1621 gccgtcaacc aagctctgat agagttggtc aagaccaatg cggagcatat acgcccggag
     1681 ccgcggcgat cctgcaagct ccggatgcct ccgctcgaag tagcgcgtct gctgctccat
     1741 acaagccaac cacggcctcc agaagaagat gttggcgacc tcgtattggg aatccccgaa
     1801 catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt
     1861 ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg cagtcctcgg cccaaagcat
     1921 cagctcatcg agagcctgcg cgacggacgc actgacggtg tcgtccatca cagtttgcca
     1981 gtgatacaca tggggatcag caatcgcgca tatgaaatca cgccatgtag tgtattgacc
     2041 gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg ctaagatcgg ccgcagcgat
     2101 cgcatccatg gcctccgcga ccggctgcag aacagcgggc agttcggttt caggcaggtc
     2161 ttgcaacgtg acaccctgtg cacggcggga gatgcaatag gtcaggctct cgctaaattc
     2221 cccaatgtca agcacttccg gaatcgggag cgcggccgat gcaaagtgcc gataaacata
     2281 acgatctttg tagaaaccat cggcgcagct atttacccgc aggacatatc cacgccctcc
     2341 tacatcgaag ctgaaagcac gagattcttc gccctccgag agctgcatca ggtcggagac
     2401 gctgtcgaac ttttcgatca gaaacttctc gacagacgtc gcggtgagtt caggcttttt
     2461 cattttgatt gctagcaagc ttggtcaagc taattggctc tcagatcctc acgggcggcg
     2521 acggcggcga actctcctca gcgcggcggc ggcggcagat gagccccggc tgcgggagca
     2581 ggcggaggct tttgtccctg acactcgggg gcctcggcgg acgcagcgga gacacagcgc
     2641 gcggctcagg ctccggctcc gactcggcga cgctctagcc caagagctca atgcccgtct
     2701 ctatgctttt acgcgcgcgc ccgcccccca ccgcctaaag cagagcgccg aacagcggag
     2761 gcgggcggcg cgcgcggcgc gagcgctgac tctcacattc actcacccac gcggaagggg
     2821 cccggcgcga agaaccccgg gaagggagag gccggccgct gcgccctctg gcggattgga
     2881 ggagcggaag ccgtgctcac ggcctccccg cccccaccgc cccaggatta aggcctaggg
     2941 ggtttgggac gaccagggtc gcaccacgag gtttcgacta acacctggaa agactagctc
     3001 tggcttcaga agaacgacaa aagccggttt ctcagtgagg gaggggcctc cgaagcccgc
     3061 gagaccttaa aatgaacctg ccttccagct gcaagtgata ccccaccccc atctgggagg
     3121 taacccgacc cctagggagt cacacagtcc ctcatggcgt aatcatggtc atagctgttt
     3181 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3241 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3301 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3361 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3421 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3481 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3541 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3601 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3661 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3721 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     3781 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3841 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3901 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3961 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4021 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4081 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4141 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4201 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4261 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4321 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4381 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4441 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4501 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4561 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4621 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4681 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4741 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     4801 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4861 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4921 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4981 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5041 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5101 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5161 agggcgacac ggaa
//
