LOCUS       sequence-202815-        4885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses As crRNA and mCherry in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4885)
  AUTHORS   Toth E, Czene BC, Kulcsar PI, Krausz SL, Talas A, Nyeste A, Varga E,
            Huszar K, Weinhardt N, Ligeti Z, Borsy AE, Fodor E, Welker E
  TITLE     Mb- and FnCpf1 nucleases are active in mammalian cells: activities 
            and PAM preferences of four wild-type Cpf1 nucleases and of their 
            altered PAM specificity variants.
  JOURNAL   Nucleic Acids Res. 2018 Sep 20. pii: 5103950. doi: 
            10.1093/nar/gky815.
  PUBMED    30239882
REFERENCE   2  (bases 1 to 4885)
  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: "Nucleic 
            Acids Res. 2018 Sep 20. pii: 5103950. doi: 10.1093/nar/gky815."
COMMENT     Sequence Label: pTE4560
FEATURES             Location/Qualifiers
     source          1..4885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..255
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7..27
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     178..197
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        308..593
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        595..872
                     /label=chicken beta-actin promoter
     intron          873..1101
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1113..1122
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1119..1829
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1264..1282)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    1958..2079
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1995..2014)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2049..2068
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2209..2228
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2342..2446
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2448..2805
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2448..2639
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2471..2491)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2656..2791
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2840..3634
                     /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             2840..3634
                     /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(2894..2913)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3504..3523
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3822..3841)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3866..3913
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4242..4830
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4731..4750
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
       61 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      121 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      181 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      241 aaaggacgaa acaccgtaat ttctactctt gtagatggag acgggatccc gtctcctttt
      301 ttgtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      361 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      421 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      481 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat
      541 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
      601 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
      661 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg
      721 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg
      781 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
      841 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc
      901 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
      961 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg
     1021 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg
     1081 cctgaaatca ctttttttca ggttggaccg gtgccaccat ggtgagcaag ggcgaggagg
     1141 ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     1201 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     1261 ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
     1321 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     1381 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     1441 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga
     1501 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaaaaag accatgggct
     1561 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     1621 agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg
     1681 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct
     1741 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     1801 ccggcggcat ggacgagctg tacaagtagg atccaccgga tctagataac tgatcataat
     1861 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     1921 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     1981 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2041 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa cgcggtgaac catcacccta
     2101 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     2161 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     2221 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     2281 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt
     2341 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     2401 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag
     2461 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     2521 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     2581 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     2641 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     2701 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     2761 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac
     2821 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     2881 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     2941 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     3001 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg
     3061 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     3121 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     3181 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     3241 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     3301 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     3361 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     3421 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     3481 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     3541 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     3601 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     3661 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     3721 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     3781 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca
     3841 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt
     3901 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     3961 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     4021 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc
     4081 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4141 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4201 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4261 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4321 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4381 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4441 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4501 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4561 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4621 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4681 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4741 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4801 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4861 ctggcctttt gctggccttt tgctc
//
