LOCUS       sequence-84459-e        5139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes mCherry fused with an out-of-frame EGFP through a linker 
            containing a target for TALEN/CRISPR. Can be used as a surrogate 
            target to test the specificity of TALENs and CRISPR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5139)
  AUTHORS   Niccheri F, Pecori R, Conticello SG
  TITLE     An efficient method to enrich for knock-out and knock-in cellular 
            clones using the CRISPR/Cas9 system.
  JOURNAL   Cell Mol Life Sci. 2017 Apr 18. doi: 10.1007/s00018-017-2524-y.
  PUBMED    28421278
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: "Cell Mol 
            Life Sci. 2017 Apr 18. doi: 10.1007/s00018-017-2524-y."
COMMENT     Sequence Label: pBS SK mCherryROSAegfp
FEATURES             Location/Qualifiers
     source          1..5139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     669..685
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        828..1031
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     982..1002
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1069..1776
                     /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(1214..1232)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1482..1501
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     regulatory      1905..1914
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1911..1913,1914..1916,1917..2630)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1911..1913,1914..1916,1917..2627)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1956..1977)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2217..2236)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2564..2585
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2753..2874
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2790..2809)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2844..2863
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2898..2914)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2898..2914)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2951..2971)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2951..2969)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2990..3006)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2990..3006)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3003..3025)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3014..3030
                     /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(3038..3044,3045..3062,3063..3068))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3083..3104
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3221..3238)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3392..3980)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3472..3491)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4151..4942,4943..5011))
                     /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     4774..4793
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5012..5116)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttgattat gccaagtacg ccccctattg
      721 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      781 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
      841 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
      901 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
      961 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1021 taagcagagc tggtttagtg aaccgtcaga tccgctagcg gattcaccat ggtgagcaag
     1081 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     1141 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     1201 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     1261 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     1321 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     1381 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     1441 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     1501 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     1561 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     1621 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     1681 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     1741 cgccactcca ccggcggcat ggacgagctg tacaagaagc ttcgtctctg tcggataatc
     1801 tgcaactcca gtctttccag aagatgggcg ggagtcttct gggcaaagat ctgcgagacg
     1861 gaattctgca gtcgacggta ccgcgggccc gggatccacc ggtcgccacc atggtgagca
     1921 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1981 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2041 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2101 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     2161 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2221 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2281 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2341 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     2401 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     2461 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2521 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     2581 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgcg
     2641 actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     2701 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     2761 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2821 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaaatcga
     2881 attcctgcag cccgggggat ccactagttc tagagcggcc gccaccgcgg tggagctcca
     2941 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     3001 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     3061 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     3121 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     3181 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     3241 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3301 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3361 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3421 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3481 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3541 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3601 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3661 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3721 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3781 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     3841 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3901 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3961 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4021 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4081 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4141 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4201 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4261 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4321 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4381 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4441 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4501 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4561 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4621 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4681 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4741 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4801 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4861 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4921 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4981 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5041 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5101 aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
