LOCUS       sequence-232628-        4786 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with mScarlet-i.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4786)
  AUTHORS   Fueller J, Herbst K, Meurer M, Gubicza K, Kurtulmus B, Knopf JD, 
            Kirrmaier D, Buchmuller BC, Pereira G, Lemberg MK, Knop M
  TITLE     CRISPR-Cas12a-assisted PCR tagging of mammalian genes.
  JOURNAL   J Cell Biol. 2020 Jun 1;219(6). pii: 151766. doi: 
            10.1083/jcb.201910210.
  PUBMED    32406907
REFERENCE   2  (bases 1 to 4786)
  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: "J Cell 
            Biol."; date: "2020-06-1"; volume: "219(6). pii: 151766. doi"; 
            pages: " 10.1083/jcb.201910210"
COMMENT     Sequence Label: pMaCTag-P11
FEATURES             Location/Qualifiers
     source          1..4786
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             81..773
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(211..229)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     479..498
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    783..904
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(820..839)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     874..893
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(911..944)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        951..1267
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1118..1253
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1889..2113
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2120..2368
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2120..2140
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2291..2310
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2370..2403)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2440..2459)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2441..2459)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2546..2563)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2717..3305)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2797..2816)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3476..4267,4268..4336))
                     /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     4099..4118
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4337..4441)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4509..4527
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4565..4587)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4687..4706
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggcagt gatcaaggag ttcatgcggt
      121 tcaaggtgca catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg
      181 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
      241 tgcccttctc ctgggacatc ctgtcccctc agttcatgta cggctccagg gccttcatca
      301 agcaccccgc cgacatcccc gactactata agcagtcctt ccccgagggc ttcaagtggg
      361 agcgcgtgat gaacttcgag gacggcggcg ccgtgaccgt gacccaggac acctccctgg
      421 aggacggcac cctgatctac aaggtgaagc tccgcggcac caacttccct cctgacggcc
      481 ccgtaatgca gaagaagaca atgggctggg aagcgtccac cgagcggttg taccccgagg
      541 acggcgtgct gaagggcgac attaagatgg ccctgcgcct gaaggacggc ggccgctacc
      601 tggcggactt caagaccacc tacaaggcca agaagcccgt gcagatgccc ggcgcctaca
      661 acgtcgaccg caagttggac atcacctccc acaacgagga ctacaccgtg gtggaacagt
      721 acgaacgctc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtgaacta
      781 gtaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
      841 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
      901 cttagtcgat ataacttcgt atagcataca ttatacgaag ttatgtcgac ggtgtggaaa
      961 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     1021 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     1081 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     1141 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     1201 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     1261 ttgcaaagaa ttcgccacca tgactgagta caagcctact gtgcgtttag ccaccaggga
     1321 tgatgtgcca cgagccgtga ggacactagc agccgccttt gctgactacc cagctacaag
     1381 gcacaccgtg gaccctgaca gacacattga aagagtcaca gaacttcagg agctgttttt
     1441 aactagggtg ggcctggata ttggcaaagt ctgggtggcc gatgatggag cagcagttgc
     1501 agtgtggacc accccagagt ctgttgaagc tggagctgtc tttgcagaaa ttggaccaag
     1561 aatggccgag ttatctggca gcaggctggc tgctcaacag cagatggaag gattattagc
     1621 tccccacaga cccaaggagc ctgcctggtt cctggcaaca gttggagttt ctccagacca
     1681 tcagggcaag ggcttaggat ctgctgtggt gctgcctggt gtggaagcag ctgagagagc
     1741 tggggtgcca gcatttctgg agacatcagc tcctagaaac ctgcccttct atgaacggct
     1801 gggcttcaca gtgactgctg atgtggaggt gcctgagggc cccagaacct ggtgcatgac
     1861 aagaaaacca ggagcctgac tgcagcgact gtgccttcta gttgccagcc atctgttgtt
     1921 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     1981 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     2041 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     2101 gtgggctcta tggctcgagg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     2161 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     2221 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     2281 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     2341 atatatcttg tggaaaggac gaaacaccga taacttcgta tagcatacat tatacgaagt
     2401 tatggtaccg atgcagctag cccgcggatc tgccggtctc cctatagtga gtcgtattaa
     2461 tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2521 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2581 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2641 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2701 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2761 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2821 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2881 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2941 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3001 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3061 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     3121 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     3181 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     3241 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     3301 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     3361 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     3421 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3481 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3541 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3601 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3661 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3721 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3781 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3841 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3901 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3961 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     4021 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     4081 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     4141 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     4201 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     4261 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     4321 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     4381 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     4441 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     4501 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     4561 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     4621 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     4681 actatgcggc atcagagcag attgtactga gagtgcacca tatggacata ttgtcgttag
     4741 aacgcggcta caattaatac ataaccttat gtatcataca catacg
//
