LOCUS       sequence-414814-        5182 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mScarlet insertion into the N-terminus of the 
            ACTB locus for actin visualization. To be co-transfected with sgRNA 
            plasmid px330-PITCh-ACTB Addgene #207748.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5182)
  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 5182)
  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-ACTB
FEATURES             Location/Qualifiers
     source          1..5182
                     /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             1410..2105
                     /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(1543..1561)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1811..1830
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2961..2977)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2961..2977)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2974..2996)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2985..3001
                     /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(3009..3015,3016..3033,3034..3039))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3054..3075
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3192..3209)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3363..3951)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3443..3462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4122..4913,4914..4982))
                     /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     4745..4764
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4983..5087)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5155..5173
                     /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 ccctataaaa cccagcggcg
      421 cgacgcgcca ccaccgccga gaccgcgtcc gccccgcgag cacagagcct cgcctttgcc
      481 gatccgccgc ccgtccacac ccgccgccag gtaagcccgg ccagccgacc ggggcaggcg
      541 gctcacggcc cggccgcagg cggccgcggc cccttcgccc gtgcagagcc gccgtctggg
      601 ccgcagcggg gggcgcatgg ggggggaacc ggaccgccgt ggggggcgcg ggagaagccc
      661 ctgggcctcc ggagatgggg gacaccccac gccagttcgg aggcgcgagg ccgcgctcgg
      721 gaggcgcgct ccgggggtgc cgctctcggg gcgggggcaa ccggcggggt ctttgtctga
      781 gccgggctct tgccaatggg gatcgcaggg tgggcgcggc ggagcccccg ccaggcccgg
      841 tgggggctgg ggcgccattg cgcgtgcgcg ctggtccttt gggcgctaac tgcgtgcgcg
      901 ctgggaattg gcgctaattg cgcgtgcgcg ctgggactca aggcgctaac tgcgcgtgcg
      961 ttctggggcc cggggtgccg cggcctgggc tggggcgaag gcgggctcgg ccggaagggg
     1021 tggggtcgcc gcggctcccg ggcgcttgcg cgcacttcct gcccgagccg ctggccgccc
     1081 gagggtgtgg ccgctgcgtg cgcgcgcgcc gacccggcgc tgtttgaacc gggcggaggc
     1141 ggggctggcg cccggttggg agggggttgg ggcctggctt cctgccgcgc gccgcgggga
     1201 cgcctccgac cagtgtttgc cttttatggt aataacgcgg ccggcccggc ttcctttgtc
     1261 cccaatctgg gcgcgcgccg gcgccccctg gcggcctaag gactcggcgc gccggaagtg
     1321 gccagggcgg gggcgacctc ggctcacagc gcgcccggct attctcgcaa ctgacaatgg
     1381 gcaagagcaa gaccggtagc ggcggaagca tggtgagcaa gggcgaggca gtgatcaagg
     1441 agttcatgcg gttcaaggtg cacatggagg gctccatgaa cggccacgag ttcgagatcg
     1501 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     1561 agggtggccc cctgcccttc tcctgggaca tcctgtcccc tcagttcatg tacggctcca
     1621 gggccttcac caagcacccc gccgacatcc ccgactacta taagcagtcc ttccccgagg
     1681 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgccgtgacc gtgacccagg
     1741 acacctccct ggaggacggc accctgatct acaaggtgaa gctccgcggc accaacttcc
     1801 ctcctgacgg ccccgtaatg cagaagaaga caatgggctg ggaagcgtcc accgagcggt
     1861 tgtaccccga ggacggcgtg ctgaagggcg acattaagat ggccctgcgc ctgaaggacg
     1921 gcggccgcta cctggcggac ttcaagacca cctacaaggc caagaagccc gtgcagatgc
     1981 ccggcgccta caacgtcgac cgcaagttgg acatcacctc ccacaacgag gactacaccg
     2041 tggtggaaca gtacgaacgc tccgagggcc gccactccac cggcggcatg gacgagctgt
     2101 acaagggagg ttccggagga agcggaggat ctggcggtac cgatgatgat atcgcagcgc
     2161 tcgtcgtcga caacggctcc ggcatgtgca aggccggctt cgcgggcgac gatgcccccc
     2221 gggccgtctt cccctccatc gtggggcgcc ccaggcacca ggtaggggag ctggctgggt
     2281 ggggcagccc cgggagcggg cgggaggcaa gggcgctttc tctgcacagg agcctcccgg
     2341 tttccggggt gggggctgcg cccgtgctca gggcttcttg tcctttcctt cccagggcgt
     2401 gatggtgggc atgggtcaga aggattccta tgtgggcgac gaggcccaga gcaagagagg
     2461 catcctcacc ctgaagtacc ccatcgagca cggcatcgtc accaactggg acgacatgga
     2521 gaaaatctgg caccacacct tctacaatga gctgcgtgtg gctcccgagg agcaccccgt
     2581 gctgctgacc gaggcccccc tgaaccccaa ggccaaccgc gagaagatga cccaggtgag
     2641 tggcccgcta cctcttctgg tggccgcctc cctccttcct ggcctcccgg agctgcgccc
     2701 tttctcactg gttctctctt ctgccgtttt ccgtaggact ctcttctctg acctgagtct
     2761 cctttggaac tctgcaggtt ctatttgctt tttcccagat gagctctttt tctggtgttt
     2821 gtctctctga ctaggtgtct aagacagtgt tgtgggtgta ggtactaaca ctggctcgtg
     2881 tgacaaggcc atgaggctgg tgtaaagcgg ccttggagtg tgtattaagt aggcgcacag
     2941 taaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3001 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     3061 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3121 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3181 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3241 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3301 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3361 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3421 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3481 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3541 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3601 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3661 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3721 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3781 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3841 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3901 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3961 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4021 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4081 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4141 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4201 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4261 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4321 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4381 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4441 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4501 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4561 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4621 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4681 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4741 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4801 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4861 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4921 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4981 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5041 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5101 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     5161 cgtatcacga ggccctttcg tc
//
