LOCUS       sequence-228152-        4579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with sfGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4579)
  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 4579)
  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-Z06
FEATURES             Location/Qualifiers
     source          1..4579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    784..905
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(821..840)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     875..894
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(912..945)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        952..1268
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1119..1254
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1275..1284
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1281..1655
                     /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    1665..1889
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1896..2144
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1896..1916
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2067..2086
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2146..2179)
                     /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(2216..2235)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2217..2235)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2322..2339)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2493..3081)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2573..2592)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3252..4043,4044..4112))
                     /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     3875..3894
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4113..4217)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4285..4303
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4341..4363)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4463..4482
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4563..4579,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4563..4579,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccagcaagg gcgaggagct gttcaccggc gtggtgccca tcctggtgga gctggacggc
      121 gacgtgaacg gccacaagtt cagcgtgcgc ggcgagggcg agggcgacgc caccaacggc
      181 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctg
      241 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcgc
      301 cacgacttct tcaagagcgc catgcccgag ggctacgtgc aggagcgcac catcagcttc
      361 aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
      421 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct gggccacaag
      481 ctggagtaca acttcaacag ccacaacgtg tacatcaccg ccgacaagca gaagaacggc
      541 atcaaggcca acttcaagat ccgccacaac gtggaggacg gcagcgtgca gctggccgac
      601 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
      661 ctgagcaccc agagccgcct gagcaaggac cccaacgaga agcgcgacca catggtgctg
      721 ctggagttcg tgaccgccgc cggcatcacc cacggcatgg acgagctgta caagtgaact
      781 agtaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
      841 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
      901 tcttagtcga tataacttcg tatagcatac attatacgaa gttatgtcga cggtgtggaa
      961 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     1021 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     1081 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     1141 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     1201 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     1261 tttgcaaaga attcgccacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc
     1321 gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg
     1381 tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc
     1441 aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc
     1501 tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca
     1561 tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca
     1621 actgcgtgca cttcgtggcc gaggagcagg actgactgca gcgactgtgc cttctagttg
     1681 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     1741 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     1801 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     1861 gcatgctggg gatgcggtgg gctctatggc tcgaggaggg cctatttccc atgattcctt
     1921 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     1981 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     2041 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     2101 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgataac ttcgtatagc
     2161 atacattata cgaagttatg gtaccgatgc agctagcccg cggatctgcc ggtctcccta
     2221 tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat cggccaacgc
     2281 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     2341 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     2401 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     2461 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2521 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2581 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2641 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt
     2701 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2761 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     2821 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     2881 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     2941 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3001 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3061 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3121 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3181 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     3241 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     3301 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     3361 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     3421 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     3481 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3541 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     3601 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3661 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3721 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     3781 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     3841 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     3901 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     3961 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4021 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4081 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4141 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     4201 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     4261 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt
     4321 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     4381 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     4441 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
     4501 gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat catacacata
     4561 cgatttaggt gacactata
//
