LOCUS       sequence-360754-        5158 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor cassette plasmid for high-throughput tagging, encoding an 
            mNG2(11) synthetic exon in frame 2, 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_F2
FEATURES             Location/Qualifiers
     source          1..5158
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(723..750)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(842..928)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1081..1098
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             1462..2256
                     /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             1462..2256
                     /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(1516..1535)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2126..2145
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        2427..2638
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2658..2667
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2664..3062
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3179..3260
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3216..3235)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    3469..3502
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             3523..3540
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4725..4747)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4833..4973
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4888..4907
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4988..5005)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagcgga tacatatttg aatgtattta gaaaaataaa caaaagagtt tgtagaaacg
      721 caaaaaggcc atccgtcagg atggccttct gcttaatttg atgcctggca gtttatggcg
      781 ggcgtcctgc ccgccaccct ccgggccgtt gcttcgcaac gttcaaatcc gctcccggcg
      841 gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt
      901 ctttcgactg agcctttcgt tttatttgat gcctggcagt tccctactct cgcatgggga
      961 gaccccacac taccatcggc gctacggcgt ttcacttctg agttcggcat ggggtcaggt
     1021 gggaccaccg cgctactgcc gccaggcaaa ttctgtttta tcagaccgct tctgcgttct
     1081 gatttaatct gtatcaggct gaaaatcttc tctcatccgc caaaacagcc aagctggaga
     1141 ccgtttggcc ccccctcgag cgcgtagaaa gccagtccgc agaaacggtg ctgaccccgg
     1201 atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag
     1261 gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc
     1321 gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac
     1381 tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagctc tgatcaagag
     1441 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     1501 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     1561 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     1621 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg
     1681 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     1741 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     1801 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     1861 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     1921 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     1981 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     2041 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     2101 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     2161 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     2221 atcgccttct atcgccttct tgacgagttc ttctgaatta ttaactcgag atccactaga
     2281 gtgtggcggc cgcattctta taatcagcat catgatgtgg taccacatca tgatgctgat
     2341 tacccccaac tgagagaact caaaggttac cccagttggg gcgcctcacg aaatcgattc
     2401 gctcttaggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
     2461 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg
     2521 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
     2581 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt
     2641 gtcgtgacgc gggatccgcc accatggcca agcctttgtc tcaagaagaa tccaccctca
     2701 ttgaaagagc aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca
     2761 gcgcagctct ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg
     2821 ggggaccttg tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc
     2881 tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt
     2941 gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg
     3001 gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct
     3061 aaatctccag aggatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     3121 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     3181 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     3241 taaagcattt ttttcactgc accaatactt acgaacttcc actacttcct cctccgcttg
     3301 agccacttcc ggatcccatc atatcggtaa aggccttttg ccactccttg aagttgagct
     3361 cggtcgaact gccgccaccc gagcctccac ctgaacttcc agagccacca ctggagagag
     3421 gagaagagag gagattagaa gagcgaatcg atttcgtgag gcactagtcg cgcccgggga
     3481 gcccaagggc acgccctggc accgcggccg ctgagtctcg accatcatca tcatcatcat
     3541 tgagtttatc tgggataaca gggtaatgtc atctagggat aacagggtat gtcatctggg
     3601 ataacagggt aatgtatcta gggataacag ggtaatgtca tctgggataa cagggtaatg
     3661 tcatctaggg ataacagggt atgtcatctg ggataacagg gtaatgtatc tagggataac
     3721 agggtaatgt catctgggat aacagggtaa tgtcatctag ggataacagg gtatgtcatc
     3781 tgggataaca gggtaatgta tctagggata acagggtaat gtcatctggg ataacagggt
     3841 aatgtcatct agggataaca gggtatgtca tctgggataa cagggtaatg tatctaggga
     3901 taacagggta atgtcatctg ggataacagg gtaatgtcat ctagggataa cagggtatgt
     3961 catctgggat aacagggtaa tgtatctagg gataacaggg taatgtcatc tgggataaca
     4021 gggtaatgtc atctagggat aacagggtat gtcatctggg ataacagggt aatgtcatct
     4081 agggataaca gggtatgtca tctgggataa cagggtaatg tatctaggga taacagggta
     4141 atgtcatctg ggataacagg gtaatgtcat ctagggataa cagggtatgt catctgggat
     4201 aacagggtaa tgtatctagg gataacaggg taatgtcaaa cggtctccag cttggctgtt
     4261 ttggcggatg agagaagaat cgccagcgtc gcacagcatg tttgcttgtc gccgtcgcgt
     4321 ctgtcacatc ttttccgcca gcagttaggg attagcgtct taagctggcg cgaggaccaa
     4381 cgtatcagcc aggcgaagct gcttttgagc accacccgga tgcctatcgc caccgtcggt
     4441 cgcaatgttg gttttgacga tcaactctat ttctcgcggg tatttaaaaa atgcaccggg
     4501 gccagcccga gcgagttccg tgccggttgt gaagaaaaag tgaatgatgt agccgtcaag
     4561 ttgtcataat tggtaacgaa tcagacaatt gacggcttga cggagtagca tagggtttgc
     4621 agaatccctg cttcgtccat ttgacaggca cattatgcat gccgcttcgc cttcgcgcgc
     4681 gaattgatct gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     4741 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     4801 ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat
     4861 agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     4921 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt
     4981 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5041 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5101 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtt
//
