LOCUS       sequence-232636-        4756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with mKeima8.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4756)
  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 4756)
  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-P18
FEATURES             Location/Qualifiers
     source          1..4756
                     /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             join(81..83,84..743)
                     /codon_start=1
                     /product="human codon-optimized monomeric red fluorescent 
                     protein with a large Stokes shift (Guan et al. 2015)"
                     /label=hmKeima8.5
                     /translation="VSVIAEHMTYKVYMSGTVNGHYFEVEGDGEGKPYEGEQTVKLTVT
                     KGGPLPFAWHILSPQLQYGSIPFTKYPADIPDYFKQSFPEGYTWVRSMNFEDGAVCTVS
                     NDSSIRGNCFIYNVKISGENFPPNGPVMQKKTQGWEPSTERMFARDGMLIGNDHMALKL
                     EGGGHYLCEFKSAYKARKPVRMPGLYEIDRKLDVTSHNRDYTSVEQREIAIARHPLLG"
     polyA_signal    753..874
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(790..809)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     844..863
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(881..914)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        921..1237
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1088..1223
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1859..2083
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2090..2338
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2090..2110
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2261..2280
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2340..2373)
                     /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(2410..2429)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2411..2429)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2516..2533)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2687..3275)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2767..2786)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3446..4237,4238..4306))
                     /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     4069..4088
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4307..4411)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4479..4497
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4535..4557)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4657..4676
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcgtga tcgccgagca catgacctac aaggtgtaca
      121 tgagcggcac cgtgaacggc cactacttcg aggtggaggg cgacggcgag ggcaagccct
      181 acgagggcga gcagaccgtg aagctgaccg tgaccaaggg tggccccctg cccttcgcct
      241 ggcacatcct gagcccccag ctccagtacg gcagcatccc cttcaccaag taccccgccg
      301 acatccccga ctacttcaag cagagcttcc ccgagggcta cacctgggtg cgcagcatga
      361 acttcgagga cggcgccgtg tgcaccgtga gcaacgacag cagcatccgc ggcaactgct
      421 tcatctacaa cgtgaagatc agcggcgaga acttcccccc caacggcccc gtgatgcaga
      481 agaagaccca gggctgggag cccagcaccg agcgcatgtt cgcccgcgac ggaatgctga
      541 tcggcaacga ccacatggcc ctgaagctgg agggcggcgg ccactacctg tgcgagttca
      601 agagcgccta caaggcccgc aagcccgtga ggatgcccgg cctgtacgag atcgaccgca
      661 agctggacgt gaccagccac aaccgcgact acaccagcgt ggagcagcgc gagatcgcca
      721 tcgcccgcca ccccctgctg ggctgaacta gtaacttgtt tattgcagct tataatggtt
      781 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
      841 gttgtggttt gtccaaactc atcaatgtat cttagtcgat ataacttcgt atagcataca
      901 ttatacgaag ttatgtcgac ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
      961 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     1021 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     1081 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     1141 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     1201 gtgaggaggc ttttttggag gcctaggctt ttgcaaagaa ttcgccacca tgactgagta
     1261 caagcctact gtgcgtttag ccaccaggga tgatgtgcca cgagccgtga ggacactagc
     1321 agccgccttt gctgactacc cagctacaag gcacaccgtg gaccctgaca gacacattga
     1381 aagagtcaca gaacttcagg agctgttttt aactagggtg ggcctggata ttggcaaagt
     1441 ctgggtggcc gatgatggag cagcagttgc agtgtggacc accccagagt ctgttgaagc
     1501 tggagctgtc tttgcagaaa ttggaccaag aatggccgag ttatctggca gcaggctggc
     1561 tgctcaacag cagatggaag gattattagc tccccacaga cccaaggagc ctgcctggtt
     1621 cctggcaaca gttggagttt ctccagacca tcagggcaag ggcttaggat ctgctgtggt
     1681 gctgcctggt gtggaagcag ctgagagagc tggggtgcca gcatttctgg agacatcagc
     1741 tcctagaaac ctgcccttct atgaacggct gggcttcaca gtgactgctg atgtggaggt
     1801 gcctgagggc cccagaacct ggtgcatgac aagaaaacca ggagcctgac tgcagcgact
     1861 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     1921 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     1981 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2041 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggctcgagg agggcctatt
     2101 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
     2161 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     2221 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     2281 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga
     2341 taacttcgta tagcatacat tatacgaagt tatggtaccg atgcagctag cccgcggatc
     2401 tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac ctgcattaat
     2461 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     2521 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2581 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     2641 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     2701 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2761 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2821 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     2881 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     2941 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3001 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3061 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3121 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3181 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3241 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3301 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3361 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3421 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3481 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3541 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3601 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3661 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3721 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3781 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3841 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     3901 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     3961 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4021 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4081 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4141 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4201 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4261 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4321 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4381 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     4441 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     4501 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     4561 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     4621 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     4681 gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac ataaccttat
     4741 gtatcataca catacg
//
