LOCUS       sequence-174195-        3963 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  visualization and quantifying the out-of-frame mutations from genome
            editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3963)
  AUTHORS   Fu L, Wen L, Luu N, Shi YB
  TITLE     A simple and efficient method to visualize and quantify the 
            efficiency of chromosomal mutations from genome editing.
  JOURNAL   Sci Rep. 2016 Oct 17;6:35488. doi: 10.1038/srep35488.
  PUBMED    27748423
REFERENCE   2  (bases 1 to 3963)
  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: "Sci Rep."; 
            date: "2016-10-17"; volume: "6:35488. doi"; pages: " 
            10.1038/srep35488"
COMMENT     Sequence Label: pmCherry-GFP
FEATURES             Location/Qualifiers
     source          1..3963
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(193..906)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     914..930
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(1015..1722)
                     /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(1290..1309)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1559..1577
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1742..1758)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1742..1758)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1755..1777)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1766..1782
                     /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)."
     promoter        complement(join(1790..1796,1797..1814,1815..1820))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1835..1856
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1973..1990)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2144..2732)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2224..2243)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2903..3694,3695..3763))
                     /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     3526..3545
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3764..3868)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3936..3954
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatcat catttgtaca gttcatccat accatgcgtg atgcccgctg cggttacgaa
      241 ctccagcaga accatatgat cgcgtttctc gttcggatct ttagacagaa cgctttgcgt
      301 gctcagatag tgattgtctg gcagcagaac aggaccatca ccgattggag tgttttgctg
      361 gtagtgatca gccagctgca cgctgccatc ctccacgttg tggcgaattt taaaattcgc
      421 tttaatgcca tttttttgtt tatcggcggt gatgtaaaca ttgtggctgt taaaattgta
      481 ttccagctta tggcccagga tattgccgtc ttctttaaag tcaatgcctt tcagctcaat
      541 gcggtttacc agggtatcgc cttcaaattt cacttccgca cgcgttttgt acgtgccgtc
      601 atccttaaag gaaatcgtgc gttcctgcac atagccttcc ggcatggcgg acttgaagaa
      661 gtcatgctgc ttcatatggt ccggataacg agcaaagcac tgaacaccat aagtcagcgt
      721 cgttaccaga gtcggccaag gtaccggcag tttaccagta gtacagatga acttcagcgt
      781 cagtttacca ttagttgcgt caccttcacc ctcgccacgc acggaaaact tatgaccgtt
      841 gacatcacca tccagttcca ccagaatagg gacgacacca gtgaacagct cctcgccttt
      901 acgcatggta ccggtaaaac gacggccagt tgaatttcca cacagatccc ggcacaaact
      961 tccccatgca tctcccgaat tcgagctcgg tacccgggga tcctctagag tcgacttgta
     1021 cagctcgtcc atgccgccgg tggagtggcg gccctcggcg cgttcgtact gttccacgat
     1081 ggtgtagtcc tcgttgtggg aggtgatgtc caacttgatg ttgacgttgt aggcgccggg
     1141 cagctgcacg ggcttcttgg ccttgtaggt ggtcttgacc tcagcgtcgt agtggccgcc
     1201 gtccttcagc ttcagcctct gcttgatctc gcccttcagg gcgccgtcct cggggtacat
     1261 ccgctcggag gaggcctccc agcccatggt cttcttctgc attacggggc cgtcggaggg
     1321 gaagttggtg ccgcgcagct tcaccttgta gatgaactcg ccgtcctgca gggaggagtc
     1381 ctgggtcacg gtcaccacgc cgccgtcctc gaagttcatc acgcgctccc acttgaagcc
     1441 ctcggggaag gacagcttca agtagtcggg gatgtcggcg gggtgcttca cgtaggcctt
     1501 ggagccgtac atgaactgag gggacaggat gtcccaggcg aagggcaggg ggccaccctt
     1561 ggtcaccttc agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc
     1621 gatctcgaac tcgtggccgt tcacggagcc ctccatgtgc accttgaagc gcatgaactc
     1681 cttgatgatg gccatgttat cctcctcgcc cttgctcacc atcaagcttg gcgtaatcat
     1741 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     1801 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     1861 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     1921 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     1981 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     2041 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     2101 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2161 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2221 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2281 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2341 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2401 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2461 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2521 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2581 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2641 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     2701 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     2761 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     2821 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     2881 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     2941 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     3001 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     3061 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     3121 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     3181 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     3241 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3301 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     3361 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     3421 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     3481 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     3541 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     3601 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     3661 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     3721 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     3781 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     3841 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     3901 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     3961 gtc
//
