LOCUS       sequence-228032-        2951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with 1xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2951)
  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 2951)
  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-27
FEATURES             Location/Qualifiers
     source          1..2951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..90
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    100..221
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(137..156)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     191..210
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(228..261)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        268..516
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     268..288
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     439..458
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(518..551)
                     /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(588..607)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(589..607)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(694..711)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(865..1453)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(945..964)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1624..2415,2416..2484))
                     /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     2247..2266
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2485..2589)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2657..2675
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2713..2735)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2835..2854
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(2935..2951,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(2935..2951,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tcctacccat acgacgtccc agactacgct tgaactagta acttgtttat tgcagcttat
      121 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
      181 cattctagtt gtggtttgtc caaactcatc aatgtatctt agtcgatata acttcgtata
      241 gcatacatta tacgaagtta tgtcgaggag ggcctatttc ccatgattcc ttcatatttg
      301 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
      361 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
      421 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
      481 ttggctttat atatcttgtg gaaaggacga aacaccgata acttcgtata gcatacatta
      541 tacgaagtta tggtaccgat gcagctagcc cgcggatctg ccggtctccc tatagtgagt
      601 cgtattaatt tcgataagcc aggttaacct gcattaatga atcggccaac gcgcggggag
      661 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
      721 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
      781 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
      841 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
      901 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
      961 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     1021 gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct
     1081 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     1141 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     1201 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     1261 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     1321 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     1381 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     1441 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     1501 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     1561 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     1621 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     1681 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     1741 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     1801 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     1861 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     1921 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     1981 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     2041 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     2101 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     2161 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     2221 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     2281 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     2341 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     2401 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     2461 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     2521 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     2581 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     2641 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga
     2701 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     2761 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     2821 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tggacatatt
     2881 gtcgttagaa cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag
     2941 gtgacactat a
//
