LOCUS       sequence-232669-        4546 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with mEos4b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4546)
  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 4546)
  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-Z17
FEATURES             Location/Qualifiers
     source          1..4546
                     /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..761
                     /codon_start=1
                     /product="fixation-resistant truly monomeric green-to-red 
                     photoswitchable fluorescent protein (Paez-Segala et al., 
                     2015)"
                     /label=mEos4b
                     /note="derived from mEos2"
                     /translation="VSAIKPDMRIKLRMEGNVNGHHFVIDGDGTGKPYEGKQTMDLEVK
                     EGGPLPFAFDILTTAFHYGNRVFVKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNAR
                     NDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLL
                     EGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHAIEILSHDKDYNKVKLYEHAVAHSGLPD
                     NARR"
     polyA_signal    768..889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(805..824)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     859..878
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(896..929)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        936..1252
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1103..1238
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1259..1268
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1265..1639
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    1649..1873
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1880..2128
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1880..1900
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2051..2070
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2130..2163)
                     /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(2200..2219)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2201..2219)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2306..2323)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2477..3065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2557..2576)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3236..4027,4028..4096))
                     /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     3859..3878
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4097..4201)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4269..4287
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4325..4347)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4447..4466
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagtgcga ttaagccaga catgaggatc aaactccgta
      121 tggaaggcaa cgtaaacggg caccactttg tgatcgacgg agatggtaca ggcaagcctt
      181 atgagggaaa acagaccatg gatcttgaag tcaaagaggg cggacctctg ccttttgcct
      241 ttgatatcct gaccactgca ttccattacg gcaacagggt attcgtgaaa tatccagaca
      301 acatacaaga ctattttaag cagtcgtttc ctaaggggta ttcgtgggaa cgaagcttga
      361 ctttcgaaga cgggggcatt tgcaatgcca gaaacgacat aacaatggaa ggggacactt
      421 tctataataa agttcgattt tatggtacca actttcccgc caatggtcca gttatgcaga
      481 agaagacgct gaaatgggag ccctccactg agaaaatgta tgtgcgtgat ggagtgctga
      541 cgggtgatat tgagatggct ttgttgcttg aaggaaatgc ccattaccga tgtgacttca
      601 gaactactta caaagctaag gagaagggtg tcaagttacc aggcgcccac tttgtggacc
      661 acgccattga gattttaagc catgacaaag attacaacaa ggttaagctg tatgagcatg
      721 ctgttgctca ttctggattg cctgacaatg ccagacgatg aactagtaac ttgtttattg
      781 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
      841 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttag tcgatataac
      901 ttcgtatagc atacattata cgaagttatg tcgacggtgt ggaaagtccc caggctcccc
      961 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     1021 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     1081 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     1141 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     1201 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagaattcgc
     1261 caccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc
     1321 ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc
     1381 cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc
     1441 ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc
     1501 ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga
     1561 gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt
     1621 ggccgaggag caggactgac tgcagcgact gtgccttcta gttgccagcc atctgttgtt
     1681 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     1741 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     1801 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     1861 gtgggctcta tggctcgagg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     1921 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     1981 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     2041 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     2101 atatatcttg tggaaaggac gaaacaccga taacttcgta tagcatacat tatacgaagt
     2161 tatggtaccg atgcagctag cccgcggatc tgccggtctc cctatagtga gtcgtattaa
     2221 tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2281 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2341 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2401 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2461 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2521 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2581 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2641 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2701 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2761 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2821 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2881 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2941 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     3001 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     3061 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     3121 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     3181 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3241 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3301 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3361 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3421 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3481 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3541 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3601 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3661 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3721 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3781 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3841 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3901 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3961 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     4021 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     4081 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     4141 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     4201 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     4261 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     4321 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     4381 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     4441 actatgcggc atcagagcag attgtactga gagtgcacca tatggacata ttgtcgttag
     4501 aacgcggcta caattaatac ataaccttat gtatcataca catacg
//
