LOCUS       sequence-467888-        7361 bp    DNA     circular SYN 15-APR-2025
DEFINITION  mammalian expression (CMV promoter) of TDGa.2 (SIM mutant E321Q) 
            fused to BioID2-HA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7361)
  TITLE     The TDG Protein Environment Connects Active DNA Demethylation with 
            Chromatin and RNA Biology
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7361)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pCMV-mTDGa.2-BioID2-HA
FEATURES             Location/Qualifiers
     source          1..7361
                     /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        617..820
                     /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"
     primer_bind     865..884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        865..883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2202..2897
                     /codon_start=1
                     /product="promiscuous R40G mutant of a biotin protein 
                     ligase from Aquifex aeolicus (Kim et al., 2016)"
                     /label=BioID2
                     /translation="FKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKWL
                     SQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKVS
                     GVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISEN
                     LKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKEI
                     LSGEFSLRRS"
     CDS             2898..2924
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2954..2971)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2960..3184
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3230..3658
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3317..3336)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3527..3548
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3667..3687)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3672..4001
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3852..3987
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3914..3933
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4068..4862
                     /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(4122..4141)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4732..4751
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5036..5157
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5073..5092)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5127..5146
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5206..5222)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5206..5222)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5219..5241)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5230..5246
                     /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(5254..5260,5261..5278,5279..5284))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5299..5320
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5437..5454)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5608..6196)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5688..5707)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6367..7158,7159..7227))
                     /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     6990..7009
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7228..7332)
                     /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 aagcagagct ctctggctaa ctagagaacc
      841 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta
      901 gcatggacgc agaggccgcg cgcagctatt ctctggagca agttcaagct ttgtattcat
      961 ttccatttca acaaatgatg gcagaagttc ctaacatggc agtcacgact ggacagcagg
     1021 tgccagcagt agctcctaac atggcaaccg tgacagaaca gcaggtgccc gcagacgctc
     1081 ctgtccagga acctgcacca gaagctccaa agagaaggaa aaggaaaccc agagcagcag
     1141 agccccagga accagtggag cccaaaaaac ctgctacgtc gaagaaatcc ggcaagtcta
     1201 caaaatcaaa ggaaaagcag gagaaaatca cagacgcgtt taaagtgaaa aggaaagtgg
     1261 accgcttcaa cggcgtctct gaagctgagc ttctgaccaa gactcttcct gacattttga
     1321 ccttcaatct ggatattgtg atcattggca ttaacccggg attaatggct gcttacaaag
     1381 gacatcacta ccctgggcct ggaaatcact tctggaagtg tctgttcatg tcggggctga
     1441 gtgaggtgca gctgaatcac atggatgacc acaccttacc cggcaagtac ggcatcggat
     1501 tcaccaacat ggtggaacgg acgacgccgg gcagcaagga tctgtctagt aaagagttcc
     1561 gggaaggagg gcgcatcctg gtgcagaaac tgcagaaata tcagccacga atagcggtgt
     1621 ttaatggaaa atgtatttat gaaattttca gtaaagaagt ttttggagta aaggttaaga
     1681 acttggaatt tgggcttcaa ccccacaaga tcccagacac agaaactctg tgctacgtca
     1741 tgccgtcgtc cagcgccaga tgtgctcagt ttccccgggc ccaggacaaa gttcattact
     1801 acattaagct gaaggacttg agagaccagc tgaaaggcat tgaacgcaac accgacgttc
     1861 aggaagtgca gtatacattt gacctgcagc ttgcgcaaga ggacgcaaag aagatggctg
     1921 ttagggaaga aaagtatgat ccaggctatg aggcagctta cggcggcgcc tatggggaaa
     1981 acccatgtaa tggggaacct tgtggcattg cttcaaatgg gctaacagcc cacagtgcgg
     2041 agccgagagg agaagcgacc cccggcgatg ttccaaatgg gcagtggatg gcacagtcgt
     2101 ttgcagagca gatcccttct tttaataatt gtgggacccg agagcaggaa gaagagagcc
     2161 acgctgaccg gtcgtacgtc tccggattcg aattcggatc cttcaagaac ctgatctggc
     2221 tgaaggaggt ggacagcacc caggagagac tgaaggagtg gaacgtgagc tacggcaccg
     2281 ccctggtggc cgacagacag accaagggca gaggcggcct gggcagaaag tggctgagcc
     2341 aggagggcgg cctgtacttc agcttcctgc tgaaccccaa ggagttcgag aacctgctgc
     2401 agctgcccct ggtgctgggc ctgagcgtga gcgaggccct ggaggagatc accgagatcc
     2461 ccttcagcct gaagtggccc aacgacgtgt acttccagga gaagaaggtg agcggcgtgc
     2521 tgtgcgagct gagcaaggac aagctgatcg tgggcatcgg catcaacgtg aaccagagag
     2581 agatccccga ggagatcaag gacagagcca ccaccctgta cgagatcacc ggcaaggact
     2641 gggacagaaa ggaggtgctg ctgaaggtgc tgaagagaat cagcgagaac ctgaagaagt
     2701 tcaaggagaa gagcttcaag gagttcaagg gcaagatcga gagcaagatg ctgtacctgg
     2761 gcgaggaggt gaagctgctg ggcgagggca agatcaccgg caagctggtg ggcctgagcg
     2821 agaagggcgg cgccctgatc ctgaccgagg agggcatcaa ggagatcctg agcggcgagt
     2881 tcagcctgag aagaagctac ccttacgatg taccggatta cgcatagaag cttaagttta
     2941 aaccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     3001 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     3061 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     3121 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     3181 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     3241 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3301 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3361 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3421 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3481 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3541 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3601 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     3661 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3721 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3781 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3841 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3901 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     3961 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     4021 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     4081 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     4141 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     4201 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     4261 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     4321 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4381 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4441 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4501 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4561 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     4621 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4681 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4741 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4801 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4861 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4921 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4981 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     5041 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5101 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5161 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     5221 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5281 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5341 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5401 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5461 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5521 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5581 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5641 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5701 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5761 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5821 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5881 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5941 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6001 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6061 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6121 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6181 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6241 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6301 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6361 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6421 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6481 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6541 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6601 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6661 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6721 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6781 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6841 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6901 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6961 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7021 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7081 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7141 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7201 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7261 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7321 aggggttccg cgcacatttc cccgaaaagt gccacctgac g
//
