LOCUS       sequence-390303-        5139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a guide RNA against a repetitive locus found in the MUC4 
            gene of human chromosome 3, with mCherry2 reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5139)
  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 5139)
  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-sgMUC4
FEATURES             Location/Qualifiers
     source          1..5139
                     /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    1867..1988
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1904..1923)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1958..1977
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1995..2450)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2132..2153)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2344..2363
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2477..2581
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2583..2940
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2583..2774
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2606..2626)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2791..2926
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2975..3769
                     /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             2975..3769
                     /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(3029..3048)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3639..3658
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3957..3976)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4001..4048
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4377..4965
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4866..4885
                     /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 caccccatgg gggcgtgacc tgtggatgct ggtttaagag
     1621 ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag
     1681 tggcaccgag tcggtgcttt ttttctcgag tactgcggcc gcggtaccgc gggcccggga
     1741 tccaccggat ctagataact gatcataatc agccatacca catttgtaga ggttttactt
     1801 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     1861 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     1921 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     1981 gtatcttaac gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     2041 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     2101 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     2161 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     2221 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     2281 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     2341 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     2401 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     2461 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2521 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2581 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     2641 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     2701 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2761 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     2821 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     2881 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     2941 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3001 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3061 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3121 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     3181 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     3241 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     3301 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     3361 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     3421 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     3481 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     3541 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     3601 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     3661 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     3721 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     3781 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     3841 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     3901 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     3961 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     4021 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     4081 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     4141 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     4201 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     4261 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4321 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4381 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4441 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4501 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4561 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4621 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4681 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4741 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4801 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4861 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4921 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4981 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5041 tcccctgatt ctgtggataa ccgtattacc gccatgcatt agttattaat agtaatcaat
     5101 tacggggtca ttagttcata gcccatatat ggagttccg
//
