LOCUS       sequence-323623-        6530 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of WTAP (Wilms tumor 1 associated protein) in human 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6530)
  AUTHORS   Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, Jia G, Yu M, Lu Z, Deng 
            X, Dai Q, Chen W, He C
  TITLE     A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine
            methylation.
  JOURNAL   Nat Chem Biol. 2014 Feb;10(2):93-5. doi: 10.1038/nchembio.1432. Epub
            2013 Dec 6.
  PUBMED    24316715
REFERENCE   2  (bases 1 to 6530)
  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: 
            "10.1038/nchembio.1432"; journalName: "Nat Chem Biol"; date: 
            "2014-02"; volume: "10"; issue: "2"; pages: "93-5"
COMMENT     Sequence Label: pcDNA3/Flag-WTAP
FEATURES             Location/Qualifiers
     source          1..6530
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    270..391
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(281..300)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     335..354
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(565..1359)
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(676..695)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1286..1305
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1426..1622)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(1434..1644)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1440..1575
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1494..1513)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     1668..1688
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1697..2125)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1807..1828)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2019..2038
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2171..2395
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2384..2401
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        2421..2439
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2421..2438
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(3655..3678)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3712..3731)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3713..3731)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3776..3979)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3805..3825)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3980..4359
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4531..4550
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4625..4729
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4730..4798,4799..5590)
                     /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(4948..4967)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5761..6349
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6250..6269
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6503..6520
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct
      241 agctagaggt cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac
      301 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
      361 tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat
      421 ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt
      481 catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg
      541 tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat
      601 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
      661 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac
      721 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
      781 caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag
      841 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
      901 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
      961 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     1021 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
     1081 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     1141 cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga
     1201 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     1261 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     1321 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     1381 ctcttgatca gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag
     1441 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc
     1501 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     1561 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg
     1621 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc ctaactgaca
     1681 cacattccac agaattaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     1741 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     1801 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     1861 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     1921 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     1981 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     2041 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     2101 cttaatgcgc cgctacaggg cgcgtgggga taccccctag agccccagct ggttctttcc
     2161 gcctcagaag ccatagagcc caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc
     2221 tcccccttgc tgtcctgccc caccccaccc cccagaatag aatgacacct actcagacaa
     2281 tgcgatgcaa tttcctcatt ttattaggaa aggacagtgg gagtggcacc ttccagggtc
     2341 aaggaaggca cgggggaggg gcaaacaaca gatggctggc aactagaagg cacagtcgag
     2401 gctgatcagc gagctctagc atttaggtga cactatagaa tagggccctc tagatgcatg
     2461 ctcgagttac aaaactgaac cctgtacatt tacacttgag tccaagccat tctgaacgtg
     2521 gcgggaaccc acagttcgat tacctttccc actcactgct ttctcctctt gagggtcatg
     2581 actggagtct gtgtcatttg attggtgtgt gagagagttt tcactgcccg tgggagagtc
     2641 tacactttca taccccgcac tgagttgatt tacgtaacca ctacctcctc tgccagttct
     2701 ctcctctgag ctgttggagg acttattacc attccctgga gaagaaggaa agtcatcttc
     2761 ggttgtgttg ccctccctgt gaaatccaga cccagacgtc ctctggcggg aggagctacc
     2821 attacttggt ccgtttgtca gacgactgca gtctttactt gtggcctctg actgttctac
     2881 aggttcagaa gctgtagtcc tgctggtact tggggcagag gcctgagact gctgctgctg
     2941 gtactgagcc aactgctggc gtgtctcctt cagctgctgc tgcagaacta gaatggtact
     3001 ctgcataccc tctacttctt catcaagctg gatgatgaag tcattcagtt catcctgact
     3061 gcttttaagc tcctcactgt atttcttctg taaagccaac tctgcttcaa gttgtgcaat
     3121 acgtccctgg gacagctgcc ttccaagctc ttgattctcc tggataagca ttcgacactt
     3181 cgccattaac tttttccctg tttggctatc aggcgtaaac ttccaggcac tcagttcatt
     3241 ttgggcttgt tccagtttgt ctttagtctg ttccagttca cctttcattt ttaggaaaaa
     3301 caagttgatc gctgggtcta ccattgttga tctcagttgg gcaacgctcg gctgctggac
     3361 ttgcttgagg tactggattt gagtagtaca ctcttgcatc tcttgttcct tggttgctag
     3421 tcgcattaca aggatgtttt ccctgcgtgc agactcctgc tgttgttgct ttagtttttc
     3481 ttcagactct cttaggccag ttacatcatt agagttaaga tctgtgtact tgccctccaa
     3541 agcttgtaca tatgcttcat attgtttcca tcttagaatt aactcatctc ttgccataac
     3601 tttgaagtct gtttcactca atcgaacctt cttgggaaga ggttcttcgt tggtcttgtc
     3661 atcgtcatcc ttgtaatcca tggatccgag ctcggtacca agcttgggtc tccctatagt
     3721 gagtcgtatt aatttcgata agccagtaag cagtgggttc tctagttagc cagagagctc
     3781 tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt
     3841 tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt
     3901 caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt
     3961 actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag
     4021 taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat
     4081 tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag
     4141 tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg
     4201 gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc
     4261 catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg
     4321 taattgatta ctattaataa ctagtcaata atcaatgtca acgcgtatat ctggcccgta
     4381 catcgcgaag cagcgcaaaa cgcctaaccc taagcagatt cttcatgcaa ttgtcggtca
     4441 agccttgcct tgttgtagct taaattttgc tcgcgcacta ctcagcgacc tccaacacac
     4501 aagcagggag cagatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     4561 accatagggg atcgggagat ctcccgatcc gtcgacgtca ggtggcactt ttcggggaaa
     4621 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4681 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     4741 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     4801 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     4861 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     4921 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     4981 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     5041 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     5101 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     5161 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     5221 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     5281 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     5341 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     5401 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     5461 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     5521 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     5581 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     5641 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5701 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5761 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5821 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5881 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5941 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6001 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6061 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6121 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6181 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6241 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6301 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6361 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     6421 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     6481 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
//
