LOCUS       sequence-84455-e        5613 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 5613)
  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 5613)
  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 mCherryROSAbsr
FEATURES             Location/Qualifiers
     source          1..5613
                     /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"
     intron          2618..2683
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             2813..2833
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3258..3339
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3295..3314)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3372..3388)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3372..3388)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3425..3445)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3425..3443)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3464..3480)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3464..3480)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3477..3499)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3488..3504
                     /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(3512..3518,3519..3536,3537..3542))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3557..3578
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3695..3712)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3866..4454)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3946..3965)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4625..5416,5417..5485))
                     /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     5248..5267
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5486..5590)
                     /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 gaattctgct ggttattgtg ctgtctcatc attttggcaa agaattaatt caagcttgga
     1921 ttcatttttg cagaaatcgg aggaagaaga atatatgaaa acatttaaca tttctcaaca
     1981 agatctagaa ttagtagaag tagcgacaga gaagattaca atgctttatg aggataataa
     2041 acatcatgtg ggagcggcaa ttcgtacgaa aacaggagaa atcatttcgg cagtacatat
     2101 tgaagcgtat ataggacgag taactgtttg tgcagaagcc attgcgattg gtagtgcagt
     2161 ttcgaatgga caaaaggatt ttgacacgat tgtagctgtt agacaccctt attctgacga
     2221 agtagataga agtattcgag tggtaagtcc ttgtggtatg tgtagggagt tgatttcaga
     2281 ctatgcacca gattgttttg tgttaataga aatgaatggc aagttagtca aaactacgat
     2341 tgaagaactc attccactca aatatacccg aaattaaaag ttttaccata ccaagcttgg
     2401 ctgctgcctg aggctggacg acctcgcgga gttctaccgg cagtgcaaat ccgtcggcat
     2461 ccaggaaacc agcagcggct atccgcgcat ccatgccccc gaactgcagg agtggggagg
     2521 cacgatggcc gctttggtcc ggatctttgt gaaggaacct tacttctgtg gtgtgacata
     2581 attggacaaa ctacctacag agatttaaag ctctaaggta aatataaaat ttttaagtgt
     2641 ataatgtgtt aaactactga ttctaattgt ttgtgtattt tagattccaa cctatggaac
     2701 tgatgaatgg gagcagtggt ggaatgcctt taatgaggaa aacctgtttt gctcagaaga
     2761 aatgccatct agtgatgatg aggctactgc tgactctcaa cattctactc ctccaaaaaa
     2821 gaagagaaag gtagaagacc ccaaggactt tccttcagaa ttgctaagtt ttttgagtca
     2881 tgctgtgttt agtaatagaa ctcttgcttg ctttgctatt tacaccacaa aggaaaaagc
     2941 tgcactgcta tacaagaaaa ttatggaaaa atattctgta acctttataa gtaggcataa
     3001 cagttataat cataacatac tgttttttct tactccacac aggcatagag tgtctgctat
     3061 taataactat gctcaaaaat tgtgtacctt tagcttttta atttgtaaag gggttaataa
     3121 ggaatatttg atgtatagtg ccttgactag agatcataat cagccatacc acatttgtag
     3181 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     3241 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3301 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt gggaattcct
     3361 gcagcccggg ggatccacta gttctagagc ggccgccacc gcggtggagc tccagctttt
     3421 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     3481 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     3541 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     3601 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3661 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3721 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3781 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3841 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3901 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3961 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4021 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4081 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4141 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4201 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4261 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     4321 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4381 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4441 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4501 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4561 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4621 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4681 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4741 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4801 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4861 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4921 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4981 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5041 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5101 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5161 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5221 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5281 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5341 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5401 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5461 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5521 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5581 gggttccgcg cacatttccc cgaaaagtgc cac
//
