LOCUS       sequence-416246-        4803 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mScarlet insertion into the N-terminus of the 
            LMNB1 locus for nuclear envelope visualization. To be co-transfected
            with sgRNA plasmid px330-PITCh-LMNB1 Addgene #207770.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4803)
  AUTHORS   Philip R, Sharma A, Matellan L, Erpf AC, Hsu WH, Tkach JM, Wyatt 
            HDM, Pelletier L
  TITLE     qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous 
            tagging.
  JOURNAL   EMBO J. 2024 Dec 12. doi: 10.1038/s44318-024-00337-5.
  PUBMED    39668248
REFERENCE   2  (bases 1 to 4803)
  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: "EMBO J. 
            2024 Dec 12. doi: 10.1038/s44318-024-00337-5."
COMMENT     Sequence Label: qTAG-N-Solo-mScarlet-LMNB1
FEATURES             Location/Qualifiers
     source          1..4803
                     /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"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1132..1827
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1265..1283)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1533..1552
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2582..2598)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2582..2598)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2595..2617)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2606..2622
                     /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(2630..2636,2637..2654,2655..2660))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2675..2696
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2813..2830)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2984..3572)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3064..3083)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3743..4534,4535..4603))
                     /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     4366..4385
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4604..4708)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4776..4794
                     /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 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cggggcggcg ggagggccct
      421 gggcccagag ccgcgcgggt gggcagtccc aggcgtcctt ccttacagcc ctgagcctgg
      481 tccgggaacc gcccagccgg gagggccgag ctgacggttg cccaagggcc agattttaaa
      541 tttacaggcc cggcccccga accgccgaag cgcgctgcct gctccccatt ggcccatggt
      601 agtcacgtgg aggcgccggg gcgtgccggc catgttgggg agtgcggcgc cgcggcccgc
      661 gccacctccg ccccccgcgg cttgcctcca gcccgcccct cccggccctc ctccccccgc
      721 ccgccgctcc gtgcagcctg agaggaaaca aagtgctgcg agcaggagac ggcggcggcg
      781 cgaaccctgc tgggcctcca ttcaccctcg tcttgcattt tcccgcgtgc gtgtgtgagt
      841 gggtgtgtgt gttttcttac aaagggtatt tcgcgatcga tcgattgatt cgtagttccc
      901 ccccgcgcgc ctttgccctt tgtgctgtaa tcgagctccc gccatcccag gtgcttctcc
      961 gttcctctaa acgccagcgt ctggacgtga gcgcaggtcg ccggtttgtg ccttcggtcc
     1021 ccgcttcgcc ccctgccgtc ccctccttat cacggtcccg ctcgcggcct cgccgccccg
     1081 ctgtctccgc cgcccgccat gggcaagagc aagaccggta gcggcggaag catggtgagc
     1141 aagggcgagg cagtgatcaa ggagttcatg cggttcaagg tgcacatgga gggctccatg
     1201 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     1261 accgccaagc tgaaggtgac caagggtggc cccctgccct tctcctggga catcctgtcc
     1321 cctcagttca tgtacggctc cagggccttc accaagcacc ccgccgacat ccccgactac
     1381 tataagcagt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     1441 ggcgccgtga ccgtgaccca ggacacctcc ctggaggacg gcaccctgat ctacaaggtg
     1501 aagctccgcg gcaccaactt ccctcctgac ggccccgtaa tgcagaagaa gacaatgggc
     1561 tgggaagcgt ccaccgagcg gttgtacccc gaggacggcg tgctgaaggg cgacattaag
     1621 atggccctgc gcctgaagga cggcggccgc tacctggcgg acttcaagac cacctacaag
     1681 gccaagaagc ccgtgcagat gcccggcgcc tacaacgtcg accgcaagtt ggacatcacc
     1741 tcccacaacg aggactacac cgtggtggaa cagtacgaac gctccgaggg ccgccactcc
     1801 accggcggca tggacgagct gtacaaggga ggttccggag gaagcggagg atctggcggt
     1861 accgcgactg cgacccccgt gccgccgcgg atgggcagcc gcgctggcgg ccccaccacg
     1921 ccgctgagcc ccacgcgcct gtcgcggctc caggagaagg aggagctgcg cgagctcaat
     1981 gaccggctgg cggtgtacat cgacaaggtg cgcagcctgg agacggagaa cagcgcgctg
     2041 cagctgcagg tgacggagcg cgaggaggtg cgcggccgtg agctcaccgg cctcaaggcg
     2101 ctctacgaga ccgagctggc cgacgcgcga cgcgcgctcg acgacacggc ccgcgagcgc
     2161 gccaagctgc agatcgagct gggcaagtgc aaggcggaac acgaccagct gctcctcaag
     2221 tgagtgctag ctggcggccg cgttagcgcc aaggaggggc gggggcgcaa ccgcggcgac
     2281 cagctcaccg ggttctgccg tggggaggga gcagaggcca ggatgcacgc gtccttctga
     2341 aggaacaggg tctcggtctc cggaaaggag aaagaatcta gagttcatag cggagcaggg
     2401 gtcgcggagg gggctcgagc tgtagcgctg gggggccgtg atgcccattt ctagattttg
     2461 gatacccgct gggacgtggt aagtgcgcgc ctgggactgc cgagaaggag ctcccgcttt
     2521 cgcactcgaa tccggggagc cggcgcggag aggcggcccc tcaaagcttg gcgtaatcat
     2581 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     2641 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     2701 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     2761 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     2821 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     2881 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     2941 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3001 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3061 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3121 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3181 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3241 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3301 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3361 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3421 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3481 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3541 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3601 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3661 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3721 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     3781 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     3841 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     3901 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     3961 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4021 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4081 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4141 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4201 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4261 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4321 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4381 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4441 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4501 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4561 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4621 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4681 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     4741 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     4801 gtc
//
