LOCUS       sequence-243723-        6132 bp    DNA     circular SYN 14-APR-2025
DEFINITION  A donor plasmid for tagging the N-terminus of human POLR2A with 
            mAID-mClover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6132)
  AUTHORS   Nagashima R, Hibino K, Ashwin SS, Babokhov M, Fujishiro S, Imai R, 
            Nozaki T, Tamura S, Tani T, Kimura H, Shribak M, Kanemaki MT, Sasai 
            M, Maeshima K
  TITLE     Single nucleosome imaging reveals loose genome chromatin networks 
            via active RNA polymerase II.
  JOURNAL   J Cell Biol. 2019 Mar 1. pii: jcb.201811090. doi: 
            10.1083/jcb.201811090.
  PUBMED    30824489
REFERENCE   2  (bases 1 to 6132)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol. 2019 Mar 1. pii: jcb.201811090. doi: 10.1083/jcb.201811090."
COMMENT     Sequence Label: mAC-POLR2A donor (Hygro)
FEATURES             Location/Qualifiers
     source          1..6132
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     677..693
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     677..693
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1255..2277
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1255..2277
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2290..2346
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2353..2556
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             join(2563..2565,2566..2568,2569..3279)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2608..2629)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3216..3237
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3898..3914)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3899..3915)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3944..3964)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3944..3962)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3983..3999)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3983..3999)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3996..4018)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4007..4023
                     /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(4031..4037,4038..4055,4056..4061))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4076..4097
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4214..4231)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4385..4973)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4465..4484)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5144..5935,5936..6004))
                     /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     5767..5786
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6005..6109)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtggc ggccgctcta gaactagtgg atcccccggg ctgcaggaat tcgattttac
      721 ccacgactct ggctcgcacc ccttgcgggc ctgcgcaggc ggccccgtag cgcaagggag
      781 ggcgggaaag gaaggggcgg ggacgcgagg gcgaatctat aagaagcgtc gttcagcgag
      841 ttcgctgctc agaagcgccg agagcgcggc cgggacggtt ggagaagaag gcggctcccg
      901 gaagggggag agacaaactg ccgtaacctc tgccgttcag gaacccggtt acttatttat
      961 tcgttaccct ttttcttctt cctcccccaa aaaccttttc cttttccctt cttttttttt
     1021 cctttttggg agctgaaaaa tttccggtaa gggaaagaag ggctcctttc gctccttatt
     1081 tccccgcctc tttccctccc ccaccttccc ctcctccggc tttttcctcc caactcgggg
     1141 aggtccttcc cggtggccgc cctgacgagg tctgagcacc taggcggagg cggcgcaggc
     1201 tttttgtagt gaggtttgcg cctgcgcagc gcgcctgcct ccgccatcgc caccatgaaa
     1261 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     1321 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     1381 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     1441 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     1501 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     1561 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     1621 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     1681 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     1741 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     1801 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     1861 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     1921 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     1981 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     2041 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     2101 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     2161 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     2221 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatca
     2281 ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct
     2341 ggacctagat ctaaggagaa gagtgcttgt cctaaagatc cagccaaacc tccggccaag
     2401 gcacaagttg tgggatggcc accggtgaga tcataccgga agaacgtgat ggtttcctgc
     2461 caaaaatcaa gcggtggccc ggaggcggcg gcgttcgtga aggtatcaat ggacggagca
     2521 ccgtacttga ggaaaatcga tttgaggatg tataaagcta gcatggtgag caagggcgag
     2581 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     2641 aagttcagcg tccgcggcga gggcgagggc gatgccacca acggcaagct gaccctgaag
     2701 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc
     2761 tacggcgtgg cctgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     2821 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct ctttcaagga cgacggtacc
     2881 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     2941 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacttc
     3001 aacagccaca acgtctatat cacggccgac aagcagaaga acggcatcaa ggctaacttc
     3061 aagatccgcc acaacgttga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     3121 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ccatcagtcc
     3181 aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     3241 gccgccggga ttacacatgg catggacgag ctgtacaagg gcgctggtgc aggcgccgga
     3301 tccatgcacg ggggtggccc cccctcgggg gacagcgcat gcccgctgcg caccatcaag
     3361 agagtccagt tcggagtcct gagtccggat gaactggtaa gcggctctgt cctccccttc
     3421 ccccctcctc ccctggcggg cggggccgga cgggggctgc ggaaacttgg cgctttctcg
     3481 ctgcttatgg gtgacgggcc aggagcatgg ctcagcagcg ccaaggccgt gtaggcgcca
     3541 gtctcgggcc tcccagagtt atattttgca aagagttgag caggcgtagc gctttgtccg
     3601 agatgggggg ctggctggag ggtgagaagg agggagagaa ggatgactga tttcacactc
     3661 cagagttacc gacgttaaag gcgatccgac gaatcctagg gagtctttac aagtttggtt
     3721 aagaagccaa acccgggtag cttcctcctc ttctgtaata caaatgttaa ctgagcacct
     3781 gctctgttga agacacagga gactcgggtt acagaggtga acacgacaga ccaggaccct
     3841 accttcaaga gcactgtctg tgggaaaggg caagcagtaa acatgaatca agcttatcga
     3901 taccgtcgac ctcgaggggg ggcccggtac ccagcttttg ttccctttag tgagggttaa
     3961 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4021 caattccaca caacatacga gccgggagca taaagtgtaa agcctggggt gcctaatgag
     4081 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4141 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4201 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4261 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4321 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4381 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4441 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4501 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4561 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4621 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4681 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4741 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4801 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     4861 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4921 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4981 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5041 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5101 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5161 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5221 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5281 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5341 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5401 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5461 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5521 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5581 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5641 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5701 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5761 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5821 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5881 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5941 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6001 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6061 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6121 gaaaagtgcc ac
//
