LOCUS       sequence-491703-        6565 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  CRISPR/Cas9 donor plasmid to tag human RPA194 with mAID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6565)
  AUTHORS   Ide S, Imai R, Ochi H, Maeshima K
  TITLE     Transcriptional suppression of ribosomal DNA with phase separation.
  JOURNAL   Sci Adv. 2020 Oct 14;6(42). pii: 6/42/eabb5953. doi: 
            10.1126/sciadv.abb5953. Print 2020 Oct.
  PUBMED    33055158
REFERENCE   2  (bases 1 to 6565)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2020-10-14"; volume: "6(42). pii: 6/42/eabb5953. doi"; pages:
            " 10.1126/sciadv.abb5953. Print 2020 Oct"
COMMENT     Sequence Label: pGEM-mAID-RPA194
FEATURES             Location/Qualifiers
     source          1..6565
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     17..38
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(90..293)
                     /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             complement(294..350)
                     /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             complement(360..1382)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(360..1382)
                     /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"
     regulatory      2153..2162
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2157..2166
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2196..2215)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2197..2215)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2222..2239)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2222..2238)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2231..2253)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2379..2834
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2466..2485)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2676..2697
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2912..3016
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3017..3085,3086..3877)
                     /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(3235..3254)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4048..4636
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4537..4556
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4790..4807
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4924..4945
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4960..4965,4966..4983,4984..4990)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4998..5014
                     /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)."
     primer_bind     5003..5025
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5022..5038
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5022..5038
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5056..5074
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5056..5073
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(join(5932..6565,1..77,78..80,81..83))
                     /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(5974..5995)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
       61 agctcctcgc ccttgctcac catgctagct ttatacatcc tcaaatcgat tttcctcaag
      121 tacggtgctc cgtccattga taccttcacg aacgccgccg cctccgggcc accgcttgat
      181 ttttggcagg aaaccatcac gttcttccgg tatgatctca ccggtggcca tcccacaact
      241 tgtgccttgg ccggaggttt ggctggatct ttaggacaag cactcttctc cttgggaccg
      301 gggttttctt ccacgtctcc tgcttgcttt aacagagaga agttcgtggc tccggatcct
      361 tcctttgccc tcggacgagt gctggggcgt cggtttccac tatcggcgag tacttctaca
      421 cagccatcgg tccagacggc cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg
      481 gctccggatc ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg
      541 ccgtcaacca agctctgata gagttggtca agaccaatgc ggagcatata cgcccggagc
      601 cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg ctgctccata
      661 caagccaacc acggcctcca gaagaagatg ttggcgacct cgtattggga atccccgaac
      721 atcgcctcgc tccagtcaat gaccgctgtt atgcggccat tgtccgtcag gacattgttg
      781 gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc ccaaagcatc
      841 agctcatcga gagcctgcgc gacggacgca ctgacggtgt cgtccatcac agtttgccag
      901 tgatacacat ggggatcagc aatcgcgcat atgaaatcac gccatgtagt gtattgaccg
      961 attccttgcg gtccgaatgg gccgaacccg ctcgtctggc taagatcggc cgcagcgatc
     1021 gcatccatgg cctccgcgac cggctgcaga acagcgggca gttcggtttc aggcaggtct
     1081 tgcaacgtga caccctgtgc acggcgggag atgcaatagg tcaggctctc gctgaattcc
     1141 ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg caaagtgccg ataaacataa
     1201 cgatctttgt agaaaccatc ggcgcagcta tttacccgca ggacatatcc acgccctcct
     1261 acatcgaagc tgaaagcacg agattcttcg ccctccgaga gctgcatcag gtcggagacg
     1321 ctgtcgaact tttcgatcag aaacttctcg acagacgtcg cggtgagttc aggctttttc
     1381 atcctccagg tccgttttga attccgacac cccaagagac gttccactca ccacctgact
     1441 attcttaatt caacctcaag cccggagtca ccacgcgatt caacgtgcgc ttgcgcgcgg
     1501 aagcggtcgc aggaacgaca tttacgtgga tgagatcact tctacatggg gggggaacaa
     1561 atagttgtac gactctctgc tcgctctttt gttttgaaaa caatcaaaga tatataagac
     1621 atcaaaataa tcatttagga aaattcctcc gaggcaaatc gggtgtatag agcttttagc
     1681 ttccgtgacg aagtctttca aaccatgagc ttgcaacctc ccattttatg attgctttcg
     1741 tcttcttccg cttcagccaa tagcggagct gttccctgct ccccgcccac tgcgccccac
     1801 cttcctgaag gtcgaggcac gctttcccaa catgctctgc agagaaatca aagatggcgg
     1861 ttgtatctgc tgttcgctgg ctgggcctcc gcagcaggct tggccagccg ctgacgggtc
     1921 ggcgggcggg tttgtgtgaa caggcacgca gctgcaggta agagacgctt agggtatccg
     1981 cgaagaagac cgcggagtca ccttagtcct atgtttccca gctggctgtg gaagtaggca
     2041 cgatgaaatg gtttgctcat gcgcgccctt aacggtcgcg tgcccttaag accaggtacg
     2101 cggaggtggc cctttgaaat aggattcgca acgcatttat tgaattcccg cggccgccat
     2161 ggcggccggg agcatgcgac gtcgggccca attcgcccta tagtgagtcg tattacaatt
     2221 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     2281 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     2341 gcccttccca acagttgcgc agcctgaatg gcgaatggac gcgccctgta gcggcgcatt
     2401 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2461 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2521 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2581 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2641 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2701 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2761 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     2821 aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2881 cgcatcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2941 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3001 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3061 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3121 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3181 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     3241 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     3301 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     3361 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     3421 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     3481 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     3541 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     3601 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     3661 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     3721 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     3781 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     3841 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     3901 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     3961 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4021 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4081 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4141 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4201 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4261 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4321 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4381 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4441 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4501 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4561 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4621 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4681 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4741 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     4801 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     4861 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     4921 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     4981 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     5041 tgattacgcc aagctattta ggtgacacta tagaatactc aagctatgca tccaacgcgt
     5101 tgggagctct cccatatggt cgacccttgc tcaccaatgt gtcattaacg cctagcttgg
     5161 tgcctggaaa aaactggatg ctcaataaat gtttgtgagg ttatagatcg atagaggaag
     5221 agagggaggt agaagtattt gtcataaaaa taggagaact tgaagacaaa taaggttctg
     5281 gaagaaagga aagtttcaag agagtgcatt ggagaatgtc aaatgctacc ttgatcaaaa
     5341 tgaagactaa agagagttcc cttgatttaa caaccagacc gagaatatag cagggtccta
     5401 gcaaagagaa gttaacgcct gaactgacac ttgaaggaca atcattttac aaacaagagg
     5461 aaacttacta cgtgcctaat taaccatgtc aggtacacta tggttaattc cacaaatgct
     5521 tgttagttaa cgaggagact ggcagatgat aacgctgcag accaggaccc tccccaccct
     5581 tatattcccc tacctcagcg caaactcttt cccccagcac caccggcctc ttatccaggc
     5641 agtccacgcc ggcccaggaa cgttcccaga actacggctc ccagcaggac aagcgccaga
     5701 caagcctgga ctcaagagga tagactgggc aaaagcgctt ctgggaatcg tagtttaggg
     5761 cgccgacctc aagatccagg ctgggcacgc tacccgacca actccttact tgagctcttc
     5821 agccgaatac atcccgaagg aaatgccctg cagccgccgc cagggcatgt tcttggagat
     5881 caacattgaa ccgccgccac cagatccacc tccaccagat ccacctccac ccttgtacag
     5941 ctcgtccatg ccatgtgtaa tcccggcggc ggtcacgaac tccagcagga ccatgtgatc
     6001 gcgcttctcg ttggggtctt tgctcagctt ggactgatgg ctcaggtagt ggttgtcggg
     6061 cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac
     6121 gctgccgtcc tcaacgttgt ggcggatctt gaagttagcc ttgatgccgt tcttctgctt
     6181 gtcggccgtg atatagacgt tgtggctgtt gaagttgtac tccagcttgt gccccaggat
     6241 gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc
     6301 ctcgaacttc acctcggcgc gggtcttgta ggtaccgtcg tccttgaaag agatggtgcg
     6361 ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc
     6421 ggggtagcgg ctgaagcagg ccacgccgta gccgaaggtg gtcacgaggg tgggccaggg
     6481 cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt tggtggcatc
     6541 gccctcgccc tcgccgcgga cgctg
//
