LOCUS       sequence-390312-        5133 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a guide RNA against a repetitive locus found in the 
            pericentromere of human chromosome 9, with mCherry2 reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5133)
  AUTHORS   Tovini L, Johnson SC, Guscott MA, Andersen AM, Spierings DCJ, 
            Wardenaar R, Foijer F, McClelland SE
  TITLE     Targeted assembly of ectopic kinetochores to induce 
            chromosome-specific segmental aneuploidies.
  JOURNAL   EMBO J. 2023 May 15;42(10):e111587. doi: 10.15252/embj.2022111587. 
            Epub 2023 Apr 17.
  PUBMED    37063065
REFERENCE   2  (bases 1 to 5133)
  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: "EMBO J."; 
            date: "2023-05-15"; volume: "42(10):e111587. doi"; pages: " 
            10.15252/embj.2022111587. Epub 2023 Apr 17"
COMMENT     Sequence Label: p-mCherry2-sgC9cen
FEATURES             Location/Qualifiers
     source          1..5133
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      547..556
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             553..1260
                     /codon_start=1
                     /product="derivative of the monomeric red fluorescent 
                     protein mCherry"
                     /label=mCherry2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQCRTMGWEASTERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(698..716)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     promoter        1336..1584
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1336..1356
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1507..1526
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     polyA_signal    1861..1982
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1898..1917)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1952..1971
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1989..2444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2126..2147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2338..2357
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2471..2575
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2577..2934
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2577..2768
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2600..2620)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2785..2920
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2969..3763
                     /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             2969..3763
                     /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(3023..3042)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3633..3652
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3951..3970)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3995..4042
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4371..4959
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4860..4879
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccggtcgcca ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc
      601 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
      661 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
      721 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
      781 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
      841 aattgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
      901 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
      961 gacggccccg taatgcagtg tcgtaccatg ggctgggagg cctccactga gcggatgtac
     1021 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     1081 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     1141 gcctacaacg tcgacatcaa gttggacatc ctttcccaca acgaggacta caccatcgtg
     1201 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     1261 tccggactca gatctcgagc tcaagcttcg aattcatgag atatcaccca cctcccaacc
     1321 ccgaggggac ccagagaggg cctatttccc atgattcctt catatttgca tatacgatac
     1381 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa
     1441 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     1501 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     1561 atcttgtgga aaggacgaaa caccgtggaa tggaatggaa tggaagttta agagctatgc
     1621 tggaaacagc atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac
     1681 cgagtcggtg ctttttttct cgagtactgc ggccgcggta ccgcgggccc gggatccacc
     1741 ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta
     1801 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     1861 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     1921 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     1981 taacgcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     2041 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     2101 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     2161 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     2221 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     2281 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     2341 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     2401 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg
     2461 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2521 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga
     2581 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2641 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     2701 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2761 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     2821 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     2881 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagatcga
     2941 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     3001 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     3061 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     3121 cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc
     3181 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     3241 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     3301 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     3361 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     3421 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     3481 cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     3541 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     3601 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     3661 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     3721 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     3781 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     3841 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     3901 cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg
     3961 aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg
     4021 cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc
     4081 gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc
     4141 caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct
     4201 gccatagcct caggttactc atatatactt tagattgatt taaaacttca tttttaattt
     4261 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4321 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4381 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4441 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4501 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4561 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4621 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4681 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4741 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4801 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4861 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4921 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4981 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     5041 gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat caattacggg
     5101 gtcattagtt catagcccat atatggagtt ccg
//
