LOCUS       sequence-367446-        4939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid to endogenously knock out the ANKRD1 in Hek293 cells. 
            Neomycin 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 4939)
  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 4939)
  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_Neo
FEATURES             Location/Qualifiers
     source          1..4939
                     /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..2118
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             1324..2118
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1378..1397)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1988..2007
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2119..2253
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2156..2175)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2210..2229
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2942..2959)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2942..2958)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2951..2973)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3167..3186)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3286..3308
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3346..3364)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3432..3536
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3537..3605,3606..4397)
                     /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(3755..3774)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4568..4939,1..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 aaaatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     1381 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     1441 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     1501 ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     1561 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     1621 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     1681 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     1741 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     1801 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc
     1861 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     1921 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     1981 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     2041 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     2101 cttgacgagt tcttctgaaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     2161 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     2221 aaactcatca atgtatctta tcatgtctgg atcaagcgcc ccgggcggag gacctttcgg
     2281 aagccgctta gttcgcaggt gccgcacact taagtatttt gaccctttcg gtgtgttctt
     2341 gcagctggcg aacagctttt tcgctaacaa ggagtaattc attaagggtg ggggtggatt
     2401 gggttttgaa acgctccagg tccttagttt cctgtaggtc ttagaaggcc tttgtttttc
     2461 atcctcgtgg atcactctct ggaagtactc tcatgtgatg ggggagggga agcgggaggg
     2521 aatggaaatc cagaggagcc aataaaatga cctttttaga tcaacttgct ctgggtggag
     2581 ggggacattt tcactggcga ctgtcgcttc agcattcact gtcatctctt acccttttga
     2641 agttcaatct tacgtgcttt gttaactagt aaggtttttc tctggttagc atatatgaac
     2701 taatttgaaa ggaagattgt aggattggga cactcctaat tgaatttttt tttctttagc
     2761 aaaatggatc tgtacatcaa aaggatggat taaacgatga tgattttgaa acttgagtcc
     2821 acaggcaagg cccgtgagta gttaactatt tacatatcta atcgaagggt gtggtatatt
     2881 ctttctccgc cttctaccaa taaatcccac tagaggatcc ccgggtaccg agctcgaatt
     2941 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     3001 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     3061 gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc
     3121 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg
     3181 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3241 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3301 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     3361 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     3421 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3481 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     3541 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     3601 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     3661 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3721 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     3781 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3841 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3901 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     3961 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4021 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     4081 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     4141 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     4201 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     4261 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     4321 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     4381 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     4441 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     4501 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4561 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4621 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4681 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     4741 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4801 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4861 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4921 gaacgaccta caccgaact
//
