LOCUS       sequence-491701-        6106 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  CRISPR/Cas9 donor plasmid to tag human RPA194 with Halo tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6106)
  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 6106)
  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-HaloTag-RPA194
FEATURES             Location/Qualifiers
     source          1..6106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        17..35
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     17..34
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(893..1777)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             complement(1787..1843)
                     /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(1853..2449)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1933..1953)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2430..2449
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     regulatory      3220..3229
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3224..3233
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3263..3282)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3264..3282)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3289..3306)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3289..3305)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3298..3320)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3446..3901
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3533..3552)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3743..3764
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3979..4083
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4084..4152,4153..4944)
                     /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(4302..4321)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5115..5703
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5604..5623
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5857..5874
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5991..6012
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6027..6032,6033..6050,6051..6057)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6065..6081
                     /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     6070..6092
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6089..6105
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6089..6105
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atgattacgc caagctattt aggtgacact atagaatact caagctatgc atccaacgcg
       61 ttgggagctc tcccatatgg tcgacccttg ctcaccaatg tgtcattaac gcctagcttg
      121 gtgcctggaa aaaactggat gctcaataaa tgtttgtgag gttatagatc gatagaggaa
      181 gagagggagg tagaagtatt tgtcataaaa ataggagaac ttgaagacaa ataaggttct
      241 ggaagaaagg aaagtttcaa gagagtgcat tggagaatgt caaatgctac cttgatcaaa
      301 atgaagacta aagagagttc ccttgattta acaaccagac cgagaatata gcagggtcct
      361 agcaaagaga agttaacgcc tgaactgaca cttgaaggac aatcatttta caaacaagag
      421 gaaacttact acgtgcctaa ttaaccatgt caggtacact atggttaatt ccacaaatgc
      481 ttgttagtta acgaggagac tggcagatga taacgctgca gaccaggacc ctccccaccc
      541 ttatattccc ctacctcagc gcaaactctt tcccccagca ccaccggcct cttatccagg
      601 cagtccacgc cggcccagga acgttcccag aactacggct cccagcagga caagcgccag
      661 acaagcctgg actcaagagg atagactggg caaaagcgct tctgggaatc gtagtttagg
      721 gcgccgacct caagatccag gctgggcacg ctacccgacc aactccttac ttgagctctt
      781 cagccgaata catcccgaag gaaatgccct gcagccgccg ccagggcatg ttcttggaga
      841 tcaacattga accgccgcca ccagatccac ctccaccaga tccacctcca ccaccggaaa
      901 tctccagagt agacagccag cgcgcgatct cgctgccgat caggtccggg ttgtcttctt
      961 gcagcagatt cagacccggg ccgatgtcca cagccttgca gttaggcagg cttttggcca
     1021 ggcgagcggc ttcggccggt gggatcagaa cgcctggggt gccccagaac agcagcttcg
     1081 ggacagggga ctggtgcagc cagtccatgt attcttcgac cagcgcgacg atgttcgctg
     1141 gctcaccggc gattggcagc tcgtttggga agcgccacag tggctcgcgg tcaacaggat
     1201 tcaggaacgg ctcgcggtaa tggtccatct cgacttcagt cagcgggcgg acgacaccca
     1261 tcggcagcgt accctcgata aaaacgttct gatcgatgat cagcttgcgg ccgacgtcgg
     1321 tggtgcggaa ggcctggaag gtctcgcggg caaattctgg ccattcgtcc caggtcggga
     1381 tagggcggat gaactccata aatgcaatac ctttgacgcg ctctggattg cgcttggccc
     1441 agtggaaacc cagagcggag ccccagtcgt gaatgaccag gacgacctct tccagaccca
     1501 gggcttcgat gaaggcatcc atgaagcgga cgtggtcgtc gaagaaataa cccaggtctg
     1561 gtttgtcgga tttgcccata ccgatcaggt ctggagcaat gcagcgatgg gtcggtgcaa
     1621 catgcgggat gatgttgcgc cacacgtagg aggaggtcgg gttaccgtgc aggaacagca
     1681 caggggtgcc atcgcgcgga ccaacatcga cgtagtgcat gcgctcgccc aggacttcca
     1741 cataatgggg gtcgaatgga aagccagtac cgatttcgga tcccatggga ccggggtttt
     1801 cttccacgtc tcctgcttgc tttaacagag agaagttcgt ggctccggat ccggcaccgg
     1861 gcttgcgggt catgcaccag gtgcgcggtc cttcgggcac ctcgacgtcg gcggtgacgg
     1921 tgaagccgag ccgctcgtag aaggggaggt tgcggggcgc ggaggtctcc aggaaggcgg
     1981 gcaccccggc gcgctcggcc gcctccactc cggggagcac gacggcgctg cccagaccct
     2041 tgccctggtg gtcgggcgag acgccgacgg tggccaggaa ccacgcgggc tccttgggcc
     2101 ggtgcggcgc caggaggcct tccatctgtt gctgcgcggc cagccgggaa ccgctcaact
     2161 cggccatgcg cgggccgatc tcggcgaaca ccgcccccgc ttcgacgctc tccggcgtgg
     2221 tccagaccgc caccgcggcg ccgtcgtccg cgacccacac cttgccgatg tcgagcccga
     2281 cgcgcgtgag gaagagttct tgcagctcgg tgacccgctc gatgtggcgg tccgggtcga
     2341 cggtgtggcg cgtggcgggg tagtcggcga acgcggcggc gagggtgcgt acggcccggg
     2401 ggacgtcgtc gcgggtggcg aggcgcaccg tgggcttgta ctcggtcatc ctccaggtcc
     2461 gttttgaatt ccgacacccc aagagacgtt ccactcacca cctgactatt cttaattcaa
     2521 cctcaagccc ggagtcacca cgcgattcaa cgtgcgcttg cgcgcggaag cggtcgcagg
     2581 aacgacattt acgtggatga gatcacttct acatgggggg ggaacaaata gttgtacgac
     2641 tctctgctcg ctcttttgtt ttgaaaacaa tcaaagatat ataagacatc aaaataatca
     2701 tttaggaaaa ttcctccgag gcaaatcggg tgtatagagc ttttagcttc cgtgacgaag
     2761 tctttcaaac catgagcttg caacctccca ttttatgatt gctttcgtct tcttccgctt
     2821 cagccaatag cggagctgtt ccctgctccc cgcccactgc gccccacctt cctgaaggtc
     2881 gaggcacgct ttcccaacat gctctgcaga gaaatcaaag atggcggttg tatctgctgt
     2941 tcgctggctg ggcctccgca gcaggcttgg ccagccgctg acgggtcggc gggcgggttt
     3001 gtgtgaacag gcacgcagct gcaggtaaga gacgcttagg gtatccgcga agaagaccgc
     3061 ggagtcacct tagtcctatg tttcccagct ggctgtggaa gtaggcacga tgaaatggtt
     3121 tgctcatgcg cgcccttaac ggtcgcgtgc ccttaagacc aggtacgcgg aggtggccct
     3181 ttgaaatagg attcgcaacg catttattga attcccgcgg ccgccatggc ggccgggagc
     3241 atgcgacgtc gggcccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt
     3301 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     3361 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     3421 gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     3481 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     3541 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3601 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3661 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     3721 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     3781 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     3841 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     3901 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atcaggtggc
     3961 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4021 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4081 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4141 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4201 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4261 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4321 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4381 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4441 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4501 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4561 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4621 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4681 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4741 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4801 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4861 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4921 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4981 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     5041 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5101 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5161 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5221 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     5281 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5341 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5401 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5461 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5521 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5581 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5641 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5701 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5761 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5821 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5881 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     5941 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     6001 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     6061 ggaattgtga gcggataaca atttcacaca ggaaacagct atgacc
//
