LOCUS       sequence-191052-        7751 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a mutant catalytic domain of TET1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7751)
  AUTHORS   Jin C, Lu Y, Jelinek J, Liang S, Estecio MR, Barton MC, Issa JP
  TITLE     TET1 is a maintenance DNA demethylase that prevents methylation 
            spreading in differentiated cells.
  JOURNAL   Nucleic Acids Res. 2014 Jun;42(11):6956-71. doi: 10.1093/nar/gku372.
            Epub 2014 May 29.
  PUBMED    24875481
REFERENCE   2  (bases 1 to 7751)
  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: 
            "10.1093/nar/gku372"; journalName: "Nucleic Acids Res"; date: 
            "2014-06"; volume: "42"; issue: "11"; pages: "6956-71"
COMMENT     Sequence Label: pIRES-hrGFP II-mTET1_CD
FEATURES             Location/Qualifiers
     source          1..7751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      8..596
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     497..516
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        741..1120
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1121..1324
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1369..1388
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1369..1387
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3699..3770
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     misc_feature    3806..4378
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3973..3990)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4200..4219
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4384..5103
                     /codon_start=1
                     /product="humanized Renilla green fluorescent protein"
                     /label=hrGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKQILKNTGLQEIMSFKVNLEGVVNNHVFTMEGCGKGNILFGN
                     QLVQIRVTKGAPLPFAFDILSPAFQYGNRTFTKYPEDISDFFIQSFPAGFVYERTLRYE
                     DGGLVEIRSDINLIEGMFVYRVEYKGRNFPNDGPVMKKTITGLQPSFEVVYMNDGVLVG
                     QVILVYRLNSGKFYSCHMRTLMKSKGVVKDFPEYHFIQHRLEKTYVEDGGFVEQHETAI
                     AQLTSLGKPLGSLHEWV"
     primer_bind     complement(5134..5154)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5134..5152)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5172..5189)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5178..5289
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(5398..5853)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5535..5556)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5747..5766
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5880..5984
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5986..6177
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6009..6029)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6174..6308
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6357..7151
                     /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             6357..7151
                     /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(6411..6430)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7021..7040
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7339..7358)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7383..7430
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
       61 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
      121 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
      181 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
      241 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
      301 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
      361 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
      421 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
      481 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
      541 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
      601 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
      661 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccaggcgc gccgcgatgt
      721 acgggccaga tttacgcgtt gacattgatt attgactagt tattaatagt aatcaattac
      781 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      841 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      901 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      961 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     1021 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     1081 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
     1141 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     1201 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     1261 ctccgcccca ttgaagcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     1321 agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca
     1381 ctatagggag acccaagctt ctggaggccc gggctttcag ggtaccgaag aaggatccac
     1441 tagtaacggc cgccagtgtg ctggaattcg cccttaccat ggctaaagat tctgaactgc
     1501 ccacctgcag ctgtcttgat cgagttatac aaaaagacaa aggcccatat tatacacacc
     1561 ttggggcagg accaagtgtt gctgctgtca gggaaatcat ggagaatagg tatggtcaaa
     1621 aaggaaacgc aataaggata gaaatagtag tgtacaccgg taaagaaggg aaaagctctc
     1681 atgggtgtcc aattgctaag tgggttttaa gaagaagcag tgatgaagaa aaagttcttt
     1741 gtttggtccg gcagcgtaca ggccaccact gtccaactgc tgtgatggtg gtgctcatca
     1801 tggtgtggga tggcatccct cttccaatgg ccgaccggct atacacagag ctcacagaga
     1861 atctaaagtc atacaatggg caccctaccg acagaagatg caccctcaat gaaaatcgta
     1921 cctgtacatg tcaaggaatt gatccagaga cttgtggagc ttcattctct tttggctgtt
     1981 catggagtat gtactttaat ggctgtaagt ttggtagaag cccaagcccc agaagattta
     2041 gaattgatcc aagctctccc ttacatgaaa aaaaccttga agataactta cagagtttgg
     2101 ctacacgatt agctccaatt tataagcagt atgctccagt agcttaccaa aatcaggtgg
     2161 aatatgaaaa tgttgcccga gaatgtcggc ttggcagcaa ggaaggtcgt cccttctctg
     2221 gggtcactgc ttgcctggac ttctgtgctc atccctacag ggccattcac aacatgaata
     2281 atggaagcac tgtggtttgt accttaactc gagaagataa ccgctctttg ggtgttattc
     2341 ctcaagatga gcagctccat gtgctacctc tttataagct ttcagacaca gatgagtttg
     2401 gctccaagga aggaatggaa gccaagatca aatctggggc catcgaggtc ctggcacccc
     2461 gccgcaaaaa aagaacgtgt ttcactcagc ctgttccccg ttctggaaag aagagggctg
     2521 cgatgatgac ggaggttctt gcacataaga taagggcagt ggaaaagaaa cctattcccc
     2581 gaatcaagcg gaagaataac tcaacaacaa caaacaacag taagccttcg tcactgccaa
     2641 ccttagggag taacactgag accgtgcaac ctgaagtaaa aagtgaaacc gaaccccatt
     2701 ttatcttaaa aagttcagac aacactaaaa cttattcgct gatgccatcc gctcctcacc
     2761 cagtgaaaga ggcatctcca ggcttctcct ggtccccgaa gactgcttca gccacaccag
     2821 ctccactgaa gaatgacgca acagcctcat gcgggttttc agaaagaagc agcactcccc
     2881 actgtacgat gccttcggga agactcagtg gtgccaatgc agctgctgct gatggccctg
     2941 gcatttcaca gcttggcgaa gtggctcctc tccccaccct gtctgctcct gtgatggagc
     3001 ccctcattaa ttctgagcct tccactggtg tgactgagcc gctaacgcct catcagccaa
     3061 accaccagcc ctccttcctc acctctcctc aagaccttgc ctcttctcca atggaagaag
     3121 atgagcagca ttctgaagca gatgagcctc catcagacga acccctatct gatgaccccc
     3181 tgtcacctgc tgaggagaaa ttgccccaca ttgatgagta ttggtcagac agtgagcaca
     3241 tctttttgga tgcaaatatt ggtggggtgg ccatcgcacc tgctcacggc tcggttttga
     3301 ttgagtgtgc ccggcgggag ctgcacgcta ccactcctgt tgagcacccc aaccgtaatc
     3361 atccaacccg cctctccctt gtcttttacc agcacaaaaa cctaaataag ccccaacatg
     3421 gttttgaact aaacaagatt aagtttgagg ctaaagaagc taagaataag aaaatgaagg
     3481 cctcagagca aaaagaccag gcagctaatg aaggtccaga acagtcctct gaagtaaatg
     3541 aattgaacca aattccttct cataaagcat taacattaac ccatgacaat gttgtcaccg
     3601 tgtcccctta tgctctcaca cacgttgcgg ggccctataa ccattgggtc tgtaggataa
     3661 gggcgaattc tgcagatatc catcacactg gcggccgcga ctacaaggat gacgatgaca
     3721 aggattacaa agacgacgat gataaggact ataaggatga tgacgacaaa taatagcaat
     3781 tcctcgacga ctgcataggg ttacccccct ctccctcccc cccccctaac gttactggcc
     3841 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc
     3901 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta
     3961 ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag
     4021 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
     4081 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg
     4141 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat
     4201 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta
     4261 tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa
     4321 acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataatg
     4381 accatggtga gcaagcagat cctgaagaac accggcctgc aggagatcat gagcttcaag
     4441 gtgaacctgg agggcgtggt gaacaaccac gtgttcacca tggagggctg cggcaagggc
     4501 aacatcctgt tcggcaacca gctggtgcag atccgcgtga ccaagggcgc ccccctgccc
     4561 ttcgccttcg acatcctgag ccccgccttc cagtacggca accgcacctt caccaagtac
     4621 cccgaggaca tcagcgactt cttcatccag agcttccccg ccggcttcgt gtacgagcgc
     4681 accctgcgct acgaggacgg cggcctggtg gagatccgca gcgacatcaa cctgatcgag
     4741 gggatgttcg tgtaccgcgt ggagtacaag ggccgcaact tccccaacga cggccccgtg
     4801 atgaagaaga ccatcaccgg cctgcagccc agcttcgagg tggtgtacat gaacgacggc
     4861 gtgctggtgg gccaggtgat cctggtgtac cgcctgaaca gcggcaagtt ctacagctgc
     4921 cacatgcgca ccctgatgaa gagcaagggc gtggtgaagg acttccccga gtaccacttc
     4981 atccagcacc gcctggagaa gacctacgtg gaggacggcg gcttcgtaga gcagcacgag
     5041 accgccatcg cccagctgac cagcctgggc aagcccctgg gcagcctgca cgagtgggtg
     5101 taagctcgag catgcatcta gagggcccta ttccctttag tgagggttaa ttgctagagc
     5161 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     5221 cgtgccttcc ttgaccctgg aaggtgccac tctcactgtc ctttcctaat aaaatgagga
     5281 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     5341 cagcaagggg gaggattggg aagacaatag caggcatgct ggggcgccgg cgcgcgtaaa
     5401 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     5461 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     5521 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     5581 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     5641 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     5701 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     5761 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     5821 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc
     5881 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5941 aataaccctg ataaatgctt caataatatt gaaaaaggaa gaatcctgag gcggaaagaa
     6001 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     6061 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     6121 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     6181 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     6241 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     6301 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     6361 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     6421 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     6481 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     6541 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     6601 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     6661 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     6721 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     6781 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagaac
     6841 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     6901 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     6961 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     7021 cgttggctac ccgtgatatt gctgaagaac ttggcggcga atgggctgac cgcttcctcg
     7081 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     7141 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     7201 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     7261 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     7321 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     7381 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     7441 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     7501 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     7561 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     7621 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     7681 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     7741 aaagatcaaa g
//
