LOCUS       sequence-228129-        4561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with mScarlet-i.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4561)
  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 4561)
  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-Z11
FEATURES             Location/Qualifiers
     source          1..4561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..756
                     /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(194..212)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     462..481
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    766..887
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(803..822)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     857..876
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(894..927)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        934..1250
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1101..1236
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1257..1266
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1263..1637
                     /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    1647..1871
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1878..2126
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1878..1898
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2049..2068
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2128..2161)
                     /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(2198..2217)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2199..2217)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2304..2321)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2475..3063)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2555..2574)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3234..4025,4026..4094))
                     /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     3857..3876
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4095..4199)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4267..4285
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4323..4345)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4445..4464
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4545..4561,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4545..4561,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagca agggcgaggc agtgatcaag gagttcatgc ggttcaaggt gcacatggag
      121 ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
      181 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt ctcctgggac
      241 atcctgtccc ctcagttcat gtacggctcc agggccttca tcaagcaccc cgccgacatc
      301 cccgactact ataagcagtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
      361 gaggacggcg gcgccgtgac cgtgacccag gacacctccc tggaggacgg caccctgatc
      421 tacaaggtga agctccgcgg caccaacttc cctcctgacg gccccgtaat gcagaagaag
      481 acaatgggct gggaagcgtc caccgagcgg ttgtaccccg aggacggcgt gctgaagggc
      541 gacattaaga tggccctgcg cctgaaggac ggcggccgct acctggcgga cttcaagacc
      601 acctacaagg ccaagaagcc cgtgcagatg cccggcgcct acaacgtcga ccgcaagttg
      661 gacatcacct cccacaacga ggactacacc gtggtggaac agtacgaacg ctccgagggc
      721 cgccactcca ccggcggcat ggacgagctg tacaagtgaa ctagtaactt gtttattgca
      781 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
      841 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttagtc gatataactt
      901 cgtatagcat acattatacg aagttatgtc gacggtgtgg aaagtcccca ggctccccag
      961 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     1021 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     1081 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     1141 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     1201 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gaattcgcca
     1261 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg
     1321 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg
     1381 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg
     1441 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg
     1501 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc
     1561 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg
     1621 ccgaggagca ggactgactg cagcgactgt gccttctagt tgccagccat ctgttgtttg
     1681 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     1741 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     1801 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     1861 gggctctatg gctcgaggag ggcctatttc ccatgattcc ttcatatttg catatacgat
     1921 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     1981 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     2041 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     2101 atatcttgtg gaaaggacga aacaccgata acttcgtata gcatacatta tacgaagtta
     2161 tggtaccgat gcagctagcc cgcggatctg ccggtctccc tatagtgagt cgtattaatt
     2221 tcgataagcc aggttaacct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2281 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     2341 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     2401 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     2461 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     2521 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     2581 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     2641 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     2701 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     2761 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     2821 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     2881 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     2941 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     3001 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     3061 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     3121 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     3181 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     3241 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3301 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     3361 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     3421 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3481 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3541 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3601 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3661 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3721 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3781 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3841 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3901 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3961 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     4021 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     4081 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     4141 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     4201 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     4261 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     4321 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     4381 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     4441 tatgcggcat cagagcagat tgtactgaga gtgcaccata tggacatatt gtcgttagaa
     4501 cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat
     4561 a
//
