LOCUS       sequence-477005-        8516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses of GFP-tagged mTet1-DSBH in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8516)
  AUTHORS   Arroyo M, Hastert FD, Zhadan A, Schelter F, Zimbelmann S, Rausch C, 
            Ludwig AK, Carell T, Cardoso MC
  TITLE     Isoform-specific and ubiquitination dependent recruitment of Tet1 to
            replicating heterochromatin modulates methylcytosine oxidation.
  JOURNAL   Nat Commun. 2022 Sep 2;13(1):5173. doi: 10.1038/s41467-022-32799-8.
  PUBMED    36056023
REFERENCE   2  (bases 1 to 8516)
  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: "Nat 
            Commun."; date: "2022-09-2"; volume: "13(1):5173. doi"; pages: " 
            10.1038/s41467-022-32799-8"
COMMENT     Sequence Label: pGFP-mTet1-DSBH  pc2335
FEATURES             Location/Qualifiers
     source          1..8516
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(88..107)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(523..542)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     642..664
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(702..720)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        788..892
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(893..961,962..1753)
                     /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(1111..1130)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1924..2512
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2413..2432
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2666..2683
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      2789..2924
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2972..3093
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2983..3002)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3037..3056
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3251..3649)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      3646..3655
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    3660..4122
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3820..3839)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     4049..4066
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(5847..5867)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(join(5874..6584,6585..6587,6588..6590))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(5874..6584,6585..6587,6588..6590))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5916..5937)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6265..6284
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6524..6545
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      6587..6596
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(6641..6660)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          6668..7684
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(6722..6744)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(7685..7961)
                     /label=chicken beta-actin promoter
     enhancer        7963..8266
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        complement(8335..8353)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8360..8377)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8360..8376)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8369..8391)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgcgccctg tagcggcgca ttgagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
       61 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      121 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      181 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      241 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac
      481 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
      541 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
      601 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
      661 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
      721 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
      781 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
      841 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
      901 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
      961 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     1021 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     1081 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     1141 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     1201 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     1261 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     1321 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     1381 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     1441 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     1501 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     1561 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     1621 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     1681 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     1741 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     1801 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     1861 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1921 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1981 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2041 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     2101 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2161 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2221 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2281 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2341 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2401 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2461 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2521 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     2581 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     2641 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     2701 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagca attagtcagc
     2761 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2821 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     2881 ctctgagcta ttccagaagt agtgaagagg cttttttgga ggcctaggct tttgcaaaaa
     2941 gctcctcgag gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc
     3001 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     3061 tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag
     3121 atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct
     3181 ttcatagaag gcggcggtgg aatcgaaatc tcgtagcacg tgtcagtcct gctcctcggc
     3241 cacgaagtgc ttagccctcc cacacataac cagagggcag caattcacga atcccaactg
     3301 ccgtcggctg tccatcactg tccttcacta tggctttgat cccaggatgc agatcgagaa
     3361 gcacctgtcg gcaccgtccg caggggctca agatgcccct gttctcattt ccgatcgcga
     3421 cgatacaagt caggttgcca gctgccgcag cagcagcagt gcccagcacc acgagttctg
     3481 cacaaggtcc cccagtaaaa tgatatacat tgacaccagt gaagatgcgg ccgtcgctag
     3541 agagagctgc gctggcgacg ctgtagtctt cagagatggg gatgctgttg attgtagccg
     3601 ttgctctttc aatgagggtg gattcttctt gagacaaagg cttggccatg gtggcaagct
     3661 tatcatcgtg tttttcaaag gaaaaccacg tccccgtggt tcggggggcc tagacgtttt
     3721 tttaacctcg actaaacaca tgtaaagcat gtgcaccgag gccccagatc agatcccata
     3781 caatggggta ccttctgggc atccttcagc cccttgttga atacgcttga ggagagccat
     3841 ttgactcttt ccacaactat ccaactcaca acgtggcact ggggttgtgc cgcctttgca
     3901 ggtgtatctt atacacgtgg cttttggccg cagaggcacc tgtcgccagg tggggggttc
     3961 cgctgcctgc aaagggtcgc tacagacgtt gtttgtcttc aagaagcttc cagaggaact
     4021 gcttccttca cgacattcaa cagaccttgc attcctttgg cgagagggga aagaccccta
     4081 ggaatgctcg tcaagaagac agggccaggt ttccgggccc tcacattgcc aaaagacggc
     4141 aatatggtgg aaaataacat atagacaaac gcacaccggc cttattccaa gcggcttcgg
     4201 ccagtaacgt tagggggggg gggggggggg gggggggggg ggggggggcg gaattcgtta
     4261 actgcggccg cttagaccca acgattgtag ggtcccgcaa catgagtgag agagtatggg
     4321 gacacggtaa caacattgtc tcgggttaag gttgatgcaa ctcgagaagg aatttggtgt
     4381 gataaattgg cttcggggga tacatcagga cactcccggt ctgcgggctt ttttttcgtt
     4441 acttttttgc atttacactt aattttgttg atatcaaaac catgattagg cttgtttagg
     4501 cttttgtgct ggtagaatac aagggacaca cgaaaaggga cccctcgttt gggggagcgc
     4561 aaagaggtgg tagcatgaag ctccttccga gcgcactcaa tgagcaccga gccgtgaatg
     4621 ggtgctatcg ccacgccacc aaaggaagga tcggcgtaga tctcctcact gtctgaccag
     4681 aattcatcaa gttgaggcaa gttatcgtcc tcggggtgct gaggctcatc cgcttcaggg
     4741 tgccgctcat cttccacctg acaggaagcc agtttctgag aattgctgag gagagggagc
     4801 tgctggtttg actggttaga agttagctgc tcagatggac tagtgagagg ctcagcatga
     4861 acgggagaat cggctgtggg aagagactgg tgaggatgag cagccacctc ggcaggctgc
     4921 accgttccag tacattcccc agcagctgca ttagcaccac tgtgtgctcc agaaggcatt
     4981 gtgcagtgga gaatactact tgtttctgca aacccacctg aggctgtttt actgtacgta
     5041 caggtagatg tttctgcgga ggaagcctgc aggggacaga agtctgtaga ttcgtctttc
     5101 actaggtgag aagtagatga ggctgatgaa aagctcttag tgttgtgtga acctggggct
     5161 ccctttgggc tggcagagat cctcttcctt ctgagaccag cagtacctga tttattgtgg
     5221 ttctgcttca ttttggccct cttcccacat cgaggaacag gctcagtaaa acgtagtcgc
     5281 ctcttcctgg ttggcccatt gacttggatg gccccagatt tgattttggc cttcatccct
     5341 tccacggagc caaattcatc agtgtctgca agccggtata gtggcaggac gtggagttgt
     5401 tcatcctcgg gacaatttgt gtcacggcca tctgctcgaa tcaacgtaca caccacggtg
     5461 cttccgttgt gcatgttgtg aatgtccttg tgagaatggg cacaaaaatc catgcaacag
     5521 gtgacaccag agaaaggacg cccctcttca tttccaagtc gacagtctcc agcaacttct
     5581 tcatattcca cctgattttg ataagcaact ggagccatct gcttgtaaag tggagctaat
     5641 actgtagcca attcttgtaa attcttttca agctgttttt cctctctgct tatgagggtc
     5701 ttgcggtctg gaatgatcca cccggttttc acgtcacttc cttcagaact cattccagta
     5761 attctcttat agtagttatg taaggggtag tttggagcaa gtctgaattt tctggggttt
     5821 tcactcctgc gatcgcctcg agatcttcct tggaagtata ggttttctcc ggacttgtac
     5881 agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga
     5941 tcgcgcttct cgttggggtc tttgctcagg gcggactggg tgctcaggta gtggttgtcg
     6001 ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc
     6061 acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc gttcttctgc
     6121 ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg
     6181 atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg
     6241 ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg
     6301 cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg
     6361 tcggggtagc ggctgaagca ctgcacgccg taggtcaggg tggtcacgag ggtgggccag
     6421 ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca
     6481 tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg
     6541 accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcaccat ggtggcgacc
     6601 ggccggtgga tccgaattct ttgccaaaat gatgagacag cacaataacc agcacgttgc
     6661 ccaggagctg taggaaaaag aagaaggcat gaacatggtt agcagaggct ctagagccgc
     6721 cggtcacacg ccagaagccg aaccccgccc tgccccgtcc cccccgaagg cagccgtccc
     6781 cccgcggaca gccccgaggc tggagaggga gaaggggacg gcggcgcggc gacgcacgaa
     6841 ggccctcccc gcccatttcc ttcctgccgg cgccgcaccg cttcgccccg cgcccgctag
     6901 agggggtgcg gcggcgcctc ccagatttcg gctccgcaca gatttgggac aaaggaagtc
     6961 cctgcgccct ctcgcacgat taccataaaa ggcaatggct gcggctcgcc gcgcctcgac
     7021 agccgccggc gctccggggg ccgccgcgcc cctcccccga gccctccccg gcccgaggcg
     7081 gccccgcccc gcccggcacc cccacctgcc gccacccccc gcccggcacg gcgagccccg
     7141 cgccacgccc cgtacggagc cccgcacccg aagccgggcc gtgctcagca actcggggag
     7201 gggggtgcag ggggggttgc agcccgaccg acgcgcccac accccctgct caccccccca
     7261 cgcacacacc ccgcacgcag cctttgttcc cctcgcagcc ccccccgcac cgcggggcac
     7321 cgcccccggc cgcgctcccc tcgcgcacac tgcggagcgc acaaagcccc gcgccgcgcc
     7381 cgcagcgctc acagccgccg ggcagcgcgg agccgcacgc ggcgctcccc acgcacacac
     7441 acacgcacgc accccccgag ccgctccccc cgcacaaagg gccctcccgg agcccctcaa
     7501 ggctttcacg cagccacaga aaagaaacaa gccgtcatta aaccaagcgc taattacagc
     7561 ccggaggaga agggccgtcc cgcccgctca cctgtgggag taacgcggtc agtcagagcc
     7621 ggggcgggcg gcgcgaggcg gcggcggagc ggggcacggg gcgaaggcag cgcgcagcga
     7681 ctcccgcccg ccgcgcgctt cgctttttat agggccgccg ccgccgccgc ctcgccataa
     7741 aaggaaactt tcggagcgcg ccgctctgat tggctgccgc cgcacctctc cgcctcgccc
     7801 cgccccgccc ctcgccccgc cccgccccgc ctggcgcgcg cccccccccc cccccgcccc
     7861 catcgctgca caaaataatt aaaaaataaa taaatacaaa attgggggtg gggagggggg
     7921 ggagatgggg agagtgaagc agaacgtggg gctcacctcg accatggtaa tagcgatgac
     7981 taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg
     8041 ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac
     8101 ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa
     8161 agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc
     8221 gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata
     8281 tgggctatga actaatgacc ccgtaattga ttactattaa taactaggac gcgtcctata
     8341 gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     8401 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     8461 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgg
//
