LOCUS       sequence-390300-        5258 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Customisable sgRNA sequence with optimised sgRNA scaffold for 
            expression under U6 promoter, with mCherry2 reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5258)
  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 5258)
  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-sgRNA (empty)
FEATURES             Location/Qualifiers
     source          1..5258
                     /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    1986..2107
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2023..2042)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2077..2096
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2114..2569)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2251..2272)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2463..2482
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2596..2700
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2702..3059
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2702..2893
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2725..2745)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2910..3045
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3094..3888
                     /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             3094..3888
                     /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(3148..3167)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3758..3777
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4076..4095)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4120..4167
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4496..5084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4985..5004
                     /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 caccggagac ggttgtaggt gagcacacaa ggtacacatg
     1621 ctaaggtatt ataccctatg accttcataa aatcaaccaa ggtcttcttt ttaataaccc
     1681 tagtatcaat acttagaatt tggatgttcc ggtttgcaac cttcgtctct gtttaagagc
     1741 tatgctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt
     1801 ggcaccgagt cggtgctttt tttctcgagt actgcggccg cggtaccgcg ggcccgggat
     1861 ccaccggatc tagataactg atcataatca gccataccac atttgtagag gttttacttg
     1921 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     1981 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     2041 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     2101 tatcttaacg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     2161 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     2221 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     2281 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     2341 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     2401 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     2461 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     2521 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact
     2581 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2641 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     2701 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     2761 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     2821 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     2881 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     2941 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     3001 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     3061 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3121 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3181 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3241 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
     3301 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3361 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3421 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3481 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3541 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3601 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     3661 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3721 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     3781 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     3841 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     3901 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     3961 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4021 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     4081 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     4141 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     4201 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     4261 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     4321 gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt
     4381 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4621 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5161 cccctgattc tgtggataac cgtattaccg ccatgcatta gttattaata gtaatcaatt
     5221 acggggtcat tagttcatag cccatatatg gagttccg
//
