LOCUS       sequence-416187-        4504 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Donor template for mScarlet insertion into the C-terminus of the 
            H3C2 locus for nuclei visualization. To be co-transfected with sgRNA
            plasmid px330-PITCh-H3C2 Addgene #207780.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4504)
  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 4504)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-C-Solo-mScarlet-H3C2
FEATURES             Location/Qualifiers
     source          1..4504
                     /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             987..1682
                     /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(1120..1138)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1388..1407
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2283..2299)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2283..2299)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2296..2318)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2307..2323
                     /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(2331..2337,2338..2355,2356..2361))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2376..2397
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2514..2531)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2685..3273)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2765..2784)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3444..4235,4236..4304))
                     /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     4067..4086
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4305..4409)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4477..4495
                     /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 cgatggacca atccaagaag
      421 ggcgcgggga tttttgaatt ttcttgggtc caatagttgg tggtctgact ctataaaaga
      481 agagtagctc tttcctttcc tccacagacg tctctgcagg caagcttttc tgtggttttg
      541 ccatggctcg tactaaacag acagctcgga aatccaccgg cggtaaagcg ccacgcaagc
      601 agctggctac caaggctgct cgcaagagcg cgccggctac cggcggcgtg aaaaagcctc
      661 accgttaccg cccgggcact gtggctctgc gcgagatccg ccgctaccaa aagtcgaccg
      721 agttgctgat tcggaagctg ccgttccagc gcctggtgcg agaaatcgcc caagacttca
      781 agaccgatct tcgcttccag agctctgcgg tgatggcgct gcaggaggct tgtgaggcct
      841 acttggtagg gctctttgag gacacaaacc tttgcgccat ccatgctaag cgagtgacta
      901 ttatgcccaa agacatccag ctcgctcggc gcattcgcgg agaaagagcg ggaggaagcg
      961 gaggatctgg cggtaccggc ggaagcatgg tgagcaaggg cgaggcagtg atcaaggagt
     1021 tcatgcggtt caaggtgcac atggagggct ccatgaacgg ccacgagttc gagatcgagg
     1081 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     1141 gtggccccct gcccttctcc tgggacatcc tgtcccctca gttcatgtac ggctccaggg
     1201 ccttcaccaa gcaccccgcc gacatccccg actactataa gcagtccttc cccgagggct
     1261 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgc cgtgaccgtg acccaggaca
     1321 cctccctgga ggacggcacc ctgatctaca aggtgaagct ccgcggcacc aacttccctc
     1381 ctgacggccc cgtaatgcag aagaagacaa tgggctggga agcgtccacc gagcggttgt
     1441 accccgagga cggcgtgctg aagggcgaca ttaagatggc cctgcgcctg aaggacggcg
     1501 gccgctacct ggcggacttc aagaccacct acaaggccaa gaagcccgtg cagatgcccg
     1561 gcgcctacaa cgtcgaccgc aagttggaca tcacctccca caacgaggac tacaccgtgg
     1621 tggaacagta cgaacgctcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     1681 agggaggttc cctcgagacc ggtaagagca agtgataaat gtaaagtcac tttttcatca
     1741 gtcttaaaac ccaaagtctc ttttcagagc cacccactta ttccaacgaa agtagctgtg
     1801 ataatttttt gttgtcttaa cagaacaaat ttctaaggac ccccccggaa agcattagac
     1861 tatggtctta aagttgatta acagaaataa cggtttggtc agtcttgcag tgtaggttat
     1921 ttctgacctt attaaggtgc tatttggaga gaagctgtgt aagtccacta tcattcaggc
     1981 ctctagcttg ctatgattag catttgttta aacaactttg taagagtaag ggaaaaatct
     2041 ggtaagtagt taactggcgc ttactaggca tttttgcaaa gctttgaaaa gattagaaaa
     2101 ttgtgtcttg cgagttccag tgtcttcctc aaaatgctta ggaagatttt ctcagctcaa
     2161 tacatagtcc cctaggtttt ctcatatatt atatctatat ctacatatat atctactgtt
     2221 aaattcattt ggctgttaac attaacctga aatttattct ggtgaagctt ggcgtaatca
     2281 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     2341 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     2401 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     2461 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2521 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2581 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2641 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2701 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2761 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2821 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2881 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2941 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3001 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3061 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3121 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3181 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3241 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3301 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3361 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3421 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3481 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3541 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3601 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3661 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3721 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3781 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3841 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3901 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3961 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4021 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4081 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4141 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4201 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4261 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4321 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4381 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     4441 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     4501 cgtc
//
