LOCUS       sequence-360738-        4747 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor cassette plasmid for high-throughput tagging, encoding an 
            mNG2(11) synthetic exon in frame 1, 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 4747)
  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 4747)
  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_F1
FEATURES             Location/Qualifiers
     source          1..4747
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(476..503)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(595..681)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     834..851
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             1215..2009
                     /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             1215..2009
                     /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(1269..1288)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1879..1898
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        2180..2391
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2411..2420
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2417..2815
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    2932..3013
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2969..2988)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    3225..3258
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             3279..3296
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4067..4089)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4175..4315
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4230..4249
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4330..4347)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(4501..4747,1..342))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4581..4600)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
       61 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
      121 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
      181 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
      241 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
      301 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
      361 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgagc
      421 ggatacatat ttgaatgtat ttagaaaaat aaacaaaaga gtttgtagaa acgcaaaaag
      481 gccatccgtc aggatggcct tctgcttaat ttgatgcctg gcagtttatg gcgggcgtcc
      541 tgcccgccac cctccgggcc gttgcttcgc aacgttcaaa tccgctcccg gcggatttgt
      601 cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtctttcga
      661 ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcatgg ggagacccca
      721 cactaccatc ggcgctacgg cgtttcactt ctgagttcgg catggggtca ggtgggacca
      781 ccgcgctact gccgccaggc aaattctgtt ttatcagacc gcttctgcgt tctgatttaa
      841 tctgtatcag gctgaaaatc ttctctcatc cgccaaaaca gccaagctgg agaccgtttg
      901 gccccccctc gagcgcgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg
      961 tcagctactg ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt
     1021 gcagtgggct tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg
     1081 aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg
     1141 ctttcttgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa gagacaggat
     1201 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     1261 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     1321 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     1381 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     1441 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     1501 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     1561 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     1621 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     1681 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     1741 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     1801 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     1861 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     1921 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     1981 tctatcgcct tcttgacgag ttcttctgaa ttattaactc gagatccact agagtgtggc
     2041 ggccgcattc ttataatcag catcatgatg tggtaccaca tcatgatgct gattaccccc
     2101 aactgagaga actcaaaggt taccccagtt ggggcgcctc acgaaatcga ttcgctctta
     2161 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
     2221 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
     2281 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
     2341 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtgtcgtga
     2401 cgcgggatcc gccaccatgg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag
     2461 agcaacggct acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc
     2521 tctctctagc gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc
     2581 ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg
     2641 tatcgtcgcg atcggaaatg agaacagggg catcttgagc ccctgcggac ggtgccgaca
     2701 ggtgcttctc gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc
     2761 gacggcagtt gggattcgtg aattgctgcc ctctggttat gtgtgggagg gctaaatctc
     2821 cagaggatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     2881 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     2941 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     3001 tttttttcac tgcaccaata cttaccagaa cttccactac ttcctcctcc gcttgagcca
     3061 cttccggatc ccatcatatc ggtaaaggcc ttttgccact ccttgaagtt gagctcggtc
     3121 gaactgccgc cacccgagcc tccacctgaa cttccagagc caccagctgg agagaggaga
     3181 agagaggaga ttagaagagc gaatcgattt cgtgaggcac tagtcgcgcc cggggagccc
     3241 aagggcacgc cctggcaccg cggccgctga gtctcgacca tcatcatcat catcattgag
     3301 tttatctggg ataacagggt aatgtcatct agggataaca gggtatgtca tctgggataa
     3361 cagggtaatg tatctaggga taacagggta atgtcatctg ggataacagg gtaatgtcat
     3421 ctagggataa cagggtatgt catctgggat aacagggtaa tgtatctagg gataacaggg
     3481 taatgtcatc tgggataaca gggtaatgtc atctagggat aacagggtat gtcatctggg
     3541 ataacagggt aatgtatcta gggataacag ggtaatgtca aacggtctcc agcttggctg
     3601 ttttggcgga tgagagaaga atcgccagcg tcgcacagca tgtttgcttg tcgccgtcgc
     3661 gtctgtcaca tcttttccgc cagcagttag ggattagcgt cttaagctgg cgcgaggacc
     3721 aacgtatcag ccaggcgaag ctgcttttga gcaccacccg gatgcctatc gccaccgtcg
     3781 gtcgcaatgt tggttttgac gatcaactct atttctcgcg ggtatttaaa aaatgcaccg
     3841 gggccagccc gagcgagttc cgtgccggtt gtgaagaaaa agtgaatgat gtagccgtca
     3901 agttgtcata attggtaacg aatcagacaa ttgacggctt gacggagtag catagggttt
     3961 gcagaatccc tgcttcgtcc atttgacagg cacattatgc atgccgcttc gccttcgcgc
     4021 gcgaattgat ctgctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     4081 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     4141 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     4201 atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     4261 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc
     4321 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4381 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4441 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4501 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4561 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4621 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4681 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4741 caagctg
//
