LOCUS       sequence-360746-        5158 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor cassette plasmid for high-throughput tagging, encoding an 
            mNG2(11) synthetic exon in frame 0, as well as a blasticidin 
            resistance gene for selection of genomic integrants..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5158)
  AUTHORS   Serebrenik YV, Sansbury SE, Kumar SS, Henao-Mejia J, Shalem O
  TITLE     Efficient and flexible tagging of endogenous genes by 
            homology-independent intron targeting.
  JOURNAL   Genome Res. 2019 Aug;29(8):1322-1328. doi: 10.1101/gr.246413.118. 
            Epub 2019 Jun 25.
  PUBMED    31239279
REFERENCE   2  (bases 1 to 5158)
  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"; doi: 
            "10.1101/gr.246413.118"; journalName: "Genome Res"; date: "2019-08";
            volume: "29"; issue: "8"; pages: "1322-1328"
COMMENT     Sequence Label: pMC_mNG2(11)_BSDminus_F0
FEATURES             Location/Qualifiers
     source          1..5158
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(343..370)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(462..548)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     701..718
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             1082..1876
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             1082..1876
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1136..1155)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1746..1765
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        2047..2258
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2278..2287
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2284..2682
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    2799..2880
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2836..2855)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    3089..3122
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             3143..3160
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4345..4367)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4453..4593
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4508..4527
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4608..4625)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(4779..5158,1..209))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4859..4878)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
       61 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
      121 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
      181 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
      241 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagcgga tacatatttg
      301 aatgtattta gaaaaataaa caaaagagtt tgtagaaacg caaaaaggcc atccgtcagg
      361 atggccttct gcttaatttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct
      421 ccgggccgtt gcttcgcaac gttcaaatcc gctcccggcg gatttgtcct actcaggaga
      481 gcgttcaccg acaaacaaca gataaaacga aaggcccagt ctttcgactg agcctttcgt
      541 tttatttgat gcctggcagt tccctactct cgcatgggga gaccccacac taccatcggc
      601 gctacggcgt ttcacttctg agttcggcat ggggtcaggt gggaccaccg cgctactgcc
      661 gccaggcaaa ttctgtttta tcagaccgct tctgcgttct gatttaatct gtatcaggct
      721 gaaaatcttc tctcatccgc caaaacagcc aagctggaga ccgtttggcc ccccctcgag
      781 cgcgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca gctactgggc
      841 tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac
      901 atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg
      961 ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc
     1021 aaggatctga tggcgcaggg gatcaagctc tgatcaagag acaggatgag gatcgtttcg
     1081 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     1141 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     1201 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     1261 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     1321 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     1381 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     1441 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     1501 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     1561 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     1621 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     1681 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     1741 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     1801 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     1861 tgacgagttc ttctgaatta ttaactcgag atccactaga gtgtggcggc cgcattctta
     1921 taatcagcat catgatgtgg taccacatca tgatgctgat tacccccaac tgagagaact
     1981 caaaggttac cccagttggg gcgcctcacg aaatcgattc gctcttaggc tccggtgccc
     2041 gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
     2101 attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
     2161 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
     2221 acgttctttt tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc gggatccgcc
     2281 accatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca
     2341 atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac
     2401 ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc
     2461 gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc
     2521 ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat
     2581 ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg
     2641 attcgtgaat tgctgccctc tggttatgtg tgggagggct aaatctccag aggatcataa
     2701 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     2761 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     2821 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     2881 accaatactt acagaacttc cactacttcc tcctccgctt gagccacttc cggatcccat
     2941 catatcggta aaggcctttt gccactcctt gaagttgagc tcggtcgaac tgccgccacc
     3001 cgagcctcca cctgaacttc cagagccacc ctggagagag gagaagagag gagattagaa
     3061 gagcgaatcg atttcgtgag gcactagtcg cgcccgggga gcccaagggc acgccctggc
     3121 accgcggccg ctgagtctcg accatcatca tcatcatcat tgagtttatc tgggataaca
     3181 gggtaatgtc atctagggat aacagggtat gtcatctggg ataacagggt aatgtatcta
     3241 gggataacag ggtaatgtca tctgggataa cagggtaatg tcatctaggg ataacagggt
     3301 atgtcatctg ggataacagg gtaatgtatc tagggataac agggtaatgt catctgggat
     3361 aacagggtaa tgtcatctag ggataacagg gtatgtcatc tgggataaca gggtaatgta
     3421 tctagggata acagggtaat gtcatctggg ataacagggt aatgtcatct agggataaca
     3481 gggtatgtca tctgggataa cagggtaatg tatctaggga taacagggta atgtcatctg
     3541 ggataacagg gtaatgtcat ctagggataa cagggtatgt catctgggat aacagggtaa
     3601 tgtatctagg gataacaggg taatgtcatc tgggataaca gggtaatgtc atctagggat
     3661 aacagggtat gtcatctggg ataacagggt aatgtcatct agggataaca gggtatgtca
     3721 tctgggataa cagggtaatg tatctaggga taacagggta atgtcatctg ggataacagg
     3781 gtaatgtcat ctagggataa cagggtatgt catctgggat aacagggtaa tgtatctagg
     3841 gataacaggg taatgtcaaa cggtctccag cttggctgtt ttggcggatg agagaagaat
     3901 cgccagcgtc gcacagcatg tttgcttgtc gccgtcgcgt ctgtcacatc ttttccgcca
     3961 gcagttaggg attagcgtct taagctggcg cgaggaccaa cgtatcagcc aggcgaagct
     4021 gcttttgagc accacccgga tgcctatcgc caccgtcggt cgcaatgttg gttttgacga
     4081 tcaactctat ttctcgcggg tatttaaaaa atgcaccggg gccagcccga gcgagttccg
     4141 tgccggttgt gaagaaaaag tgaatgatgt agccgtcaag ttgtcataat tggtaacgaa
     4201 tcagacaatt gacggcttga cggagtagca tagggtttgc agaatccctg cttcgtccat
     4261 ttgacaggca cattatgcat gccgcttcgc cttcgcgcgc gaattgatct gctgcctcgc
     4321 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     4381 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     4441 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     4501 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     4561 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     4621 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4681 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4741 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4801 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4861 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4921 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4981 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5041 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5101 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcga
//
