LOCUS       sequence-469055-        5458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses of miRFP-tagged MaSat in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5458)
  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 5458)
  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: pMaSat-miRFP670 pc3944
FEATURES             Location/Qualifiers
     source          1..5458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        764..1067
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1068..1271
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1222..1242
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1368..1387
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1368..1386
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1409..1432
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1448..1468
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1874..1883
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1880..2827
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     polyA_signal    2949..3070
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2986..3005)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3040..3059
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3077..3532)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3214..3235)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3426..3445
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3559..3663
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3665..4022
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3665..3856
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3688..3708)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3873..4008
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4057..4851
                     /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             4057..4851
                     /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(4111..4130)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4721..4740
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5039..5058)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5083..5130
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
      721 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      781 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      841 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      901 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      961 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1021 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1081 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1141 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1201 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1261 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact cagatctcga
     1321 gctctctggc taactagaga acccactgct tactggctta tcgaaattaa tacgactcac
     1381 tatagggaga cccaagcttg ccatggccga ctacaaggac gacgacgata agcggccgct
     1441 tgagcctccg aaaaagaagc gtaaggtcga gctagcaggt accgggatcc tgcaggatat
     1501 cgatctcgag gcccaggcgg ccctcgaacc caccggcgag aaaccgtacg cttgccctga
     1561 atgcggaaaa agcttctctc agtccagcaa cctcgtgagg catcagagga cccataccgg
     1621 cgagaagccc tacgcttgcc cggagtgtgg caagtcattc tccaggagcg acaatttggt
     1681 taggcatcaa cgcacacata ccggcgagaa accgtacgcg tgccctgaat gcggtaaaag
     1741 cttctctgac ccaggtcacc tcgtccgcca tcagaggacc cataccggcg agctgggtgg
     1801 cggttccgga gaaaagcccg ggaagaaaac ctcgggccag gccggccaac tagaggatcc
     1861 ccgggtaccg gtcgccacca tggtagcagg tcatgcctct ggcagccccg cattcgggac
     1921 cgcctctcat tcgaattgcg aacatgaaga gatccacctc gccggctcga tccagccgca
     1981 tggcgcgctt ctggtcgtca gcgaacatga tcatcgcgtc atccaggcca gcgccaacgc
     2041 cgcggaattt ctgaatctcg gaagcgtact cggcgttccg ctcgccgaga tcgacggcga
     2101 tctgttgatc aagatcctgc cgcatctcga tcccaccgcc gaaggcatgc cggtcgcggt
     2161 gcgctgccgg atcggcaatc cctctacgga gtactgcggt ctgatgcatc ggcctccgga
     2221 aggcgggctg atcatcgaac tcgaacgtgc cggcccgtcg atcgatctgt caggcacgct
     2281 ggcgccggcg ctggagcgga tccgcacggc gggttcactg cgcgcgctgt gcgatgacac
     2341 cgtgctgctg tttcagcagt gcaccggcta cgaccgggtg atggtgtatc gtttcgatga
     2401 gcaaggccac ggcctggtat tctccgagtg ccatgtgcct gggctcgaat cctatttcgg
     2461 caaccgctat ccgtcgtcga ctgtcccgca gatggcgcgg cagctgtacg tgcggcagcg
     2521 cgtccgcgtg ctggtcgacg tcacctatca gccggtgccg ctggagccgc ggctgtcgcc
     2581 gctgaccggg cgcgatctcg acatgtcggg ctgcttcctg cgctcgatgt cgccgtgcca
     2641 tctgcagttc ctgaaggaca tgggcgtgcg cgccaccctg gcggtgtcgc tggtggtcgg
     2701 cggcaagctg tggggcctgg ttgtctgtca ccattatctg ccgcgcttca tccgtttcga
     2761 gctgcgggcg atctgcaaac ggctcgccga aaggatcgcg acgcggatca ccgcgcttga
     2821 gagctaagcg gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac
     2881 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     2941 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3001 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3061 atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     3121 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     3181 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     3241 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     3301 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     3361 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     3421 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     3481 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
     3541 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3601 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3661 agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3721 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3781 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3841 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3901 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3961 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     4021 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     4081 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4141 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4201 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     4261 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4321 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4381 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4441 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4501 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4561 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     4621 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4681 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4741 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4801 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4861 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4921 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4981 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     5041 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     5101 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     5161 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     5221 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     5281 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     5341 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5401 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
