LOCUS       sequence-115273-        6479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes mCherry fused with out-of-frame blasticidin-S resistance 
            gene with a linker containing a target for TALEN/CRISPR. Can use as 
            a surrogate target to select cells expressing active TALEN/CRISPR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6479)
  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 6479)
  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: pBSR (mCherry-surrogateTarget-bsr)
FEATURES             Location/Qualifiers
     source          1..6479
                     /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"
     promoter        675..952
                     /label=chicken beta-actin promoter
     primer_bind     1891..1913
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             1935..2642
                     /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(2080..2098)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2348..2367
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     intron          3484..3549
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             3679..3699
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4124..4205
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4161..4180)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4238..4254)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4238..4254)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4291..4311)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4291..4309)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4330..4346)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4330..4346)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4343..4365)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4354..4370
                     /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(4378..4384,4385..4402,4403..4408))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4423..4444
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4561..4578)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4732..5320)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4812..4831)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5491..6282,6283..6351))
                     /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     6114..6133
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6352..6456)
                     /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 gaggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
      721 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
      781 gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc
      841 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
      901 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
      961 tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct
     1021 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
     1081 taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg aaagccttga
     1141 ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt gcgtgtgtgt
     1201 gtgcgtgggg agcgccgcgt gcggcccgcg ctgcccggcg gctgtgagcg ctgcgggcgc
     1261 ggcgcggggc tttgtgcgct ccgcgtgtgc gcgaggggag cgcggccggg ggcggtgccc
     1321 cgcggtgcgg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg
     1381 gtgagcaggg ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac ccccctcccc
     1441 gagttgctga gcacggcccg gcttcgggtg cggggctccg tgcggggcgt ggcgcggggc
     1501 tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg gggccgcctc
     1561 gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc ggctgtcgag
     1621 gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg cagggacttc
     1681 ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg
     1741 ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg
     1801 cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg cggggggacg
     1861 gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct
     1921 ctagcggatt caccatggtg agcaagggcg aggaggataa catggccatc atcaaggagt
     1981 tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg
     2041 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     2101 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg
     2161 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct
     2221 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     2281 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct
     2341 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt
     2401 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg
     2461 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg
     2521 gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg
     2581 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     2641 agaagcttcg tctctgtcgg ataatctgca actccagtct ttccagaaga tgggcgggag
     2701 tcttctgggc aaagatctgc gagacggaat tctgctggtt attgtgctgt ctcatcattt
     2761 tggcaaagaa ttaattcaag cttggattca tttttgcaga aatcggagga agaagaatat
     2821 atgaaaacat ttaacatttc tcaacaagat ctagaattag tagaagtagc gacagagaag
     2881 attacaatgc tttatgagga taataaacat catgtgggag cggcaattcg tacgaaaaca
     2941 ggagaaatca tttcggcagt acatattgaa gcgtatatag gacgagtaac tgtttgtgca
     3001 gaagccattg cgattggtag tgcagtttcg aatggacaaa aggattttga cacgattgta
     3061 gctgttagac acccttattc tgacgaagta gatagaagta ttcgagtggt aagtccttgt
     3121 ggtatgtgta gggagttgat ttcagactat gcaccagatt gttttgtgtt aatagaaatg
     3181 aatggcaagt tagtcaaaac tacgattgaa gaactcattc cactcaaata tacccgaaat
     3241 taaaagtttt accataccaa gcttggctgc tgcctgaggc tggacgacct cgcggagttc
     3301 taccggcagt gcaaatccgt cggcatccag gaaaccagca gcggctatcc gcgcatccat
     3361 gcccccgaac tgcaggagtg gggaggcacg atggccgctt tggtccggat ctttgtgaag
     3421 gaaccttact tctgtggtgt gacataattg gacaaactac ctacagagat ttaaagctct
     3481 aaggtaaata taaaattttt aagtgtataa tgtgttaaac tactgattct aattgtttgt
     3541 gtattttaga ttccaaccta tggaactgat gaatgggagc agtggtggaa tgcctttaat
     3601 gaggaaaacc tgttttgctc agaagaaatg ccatctagtg atgatgaggc tactgctgac
     3661 tctcaacatt ctactcctcc aaaaaagaag agaaaggtag aagaccccaa ggactttcct
     3721 tcagaattgc taagtttttt gagtcatgct gtgtttagta atagaactct tgcttgcttt
     3781 gctatttaca ccacaaagga aaaagctgca ctgctataca agaaaattat ggaaaaatat
     3841 tctgtaacct ttataagtag gcataacagt tataatcata acatactgtt ttttcttact
     3901 ccacacaggc atagagtgtc tgctattaat aactatgctc aaaaattgtg tacctttagc
     3961 tttttaattt gtaaaggggt taataaggaa tatttgatgt atagtgcctt gactagagat
     4021 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     4081 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     4141 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4201 actgcattct agttgtggga attcctgcag cccgggggat ccactagttc tagagcggcc
     4261 gccaccgcgg tggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     4321 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4381 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4441 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4501 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4561 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4621 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4681 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4741 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4801 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4861 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4921 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4981 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5041 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5101 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5161 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5221 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5281 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5341 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5401 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5461 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5521 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5581 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5641 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5701 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5761 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5821 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5881 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5941 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6001 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6061 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6121 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6181 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6241 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6301 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6361 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6421 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
