LOCUS       sequence-436805-        3675 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  golden gate cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3675)
  AUTHORS   Delhaye L, Moschonas GD, Fijalkowska D, Verhee A, De Sutter D, Van 
            de Steene T, De Meyer M, Grzesik H, Van Moortel L, De Bosscher K, 
            Jacobs T, Eyckerman S
  TITLE     Leveraging a self-cleaving peptide for tailored control in proximity
            labeling proteomics.
  JOURNAL   Cell Rep Methods. 2024 Jul 15;4(7):100818. doi: 
            10.1016/j.crmeth.2024.100818. Epub 2024 Jul 9.
  PUBMED    38986614
REFERENCE   2  (bases 1 to 3675)
  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: "Cell Rep 
            Methods."; date: "2024-07-15"; volume: "4(7):100818. doi"; pages: " 
            10.1016/j.crmeth.2024.100818. Epub 2024 Jul 9"
COMMENT     Sequence Label: pEN-X-mPGK-NeoR-SV40 polyA-Z
FEATURES             Location/Qualifiers
     source          1..3675
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     36..58
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     50..67
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     51..67
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        154..653
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(189..212)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     586..605
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             667..1461
                     /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             667..1461
                     /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(721..740)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1331..1350
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1504..1523)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1558..1577
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1686..1702)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1686..1702)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1699..1721)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(1874..2462)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1954..1973)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2598..3413)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2598..3413)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3321..3340
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3405..3424
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     3534..3550
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3534..3550
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      3551..3597
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 caataactgt gatccaccac aagcgccagg gttttcccag tcacgacgtt gtaaaacgac
       61 ggccagtcat gcataatccg cacgcatctg gaataaggaa gtgccattcc gcctgacctg
      121 aagacgtgtg acgtctccgc aaacctttct accgggtagg ggaggcgctt ttcccaaggc
      181 agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct
      241 ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc
      301 ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg
      361 cgtcgtgcag gacgtgacaa atggaagtag cacgcctcac tagtctcgtg cagatggaca
      421 gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct
      481 ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg
      541 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
      601 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctaac
      661 accaccatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
      721 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
      781 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
      841 gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
      901 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
      961 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     1021 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     1081 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     1141 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg
     1201 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     1261 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     1321 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     1381 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     1441 cttcttgacg agttcttctg aggctccact tgtttattgc agcttataat ggttacaaat
     1501 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     1561 gtttgtccaa actcatcaat gtatcttatc atgtctggct ctgtatcaga tgagacgcgt
     1621 atagtcttca ggctaggtgg aggctcagtg atgataagtc tgcgatggtg gatgcatgtg
     1681 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcagagggca caatcctatt
     1741 ccgcgctatc cgacaatctc caagacatta ggtggagttc agttcggcgt atggcatatg
     1801 tcgctggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     1861 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     1921 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     1981 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2041 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2101 aggtcgttcg ctccaagccg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2161 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2221 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2281 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     2341 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2401 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2461 aagaagatcc tttgatcttt tctacggggt ctgacgctct attcaacaaa gccgccgtcc
     2521 cgtcaagtca gcgtaaatgg gtagggggct tcaaatcgtc cgctctgcca gtgttacaac
     2581 caattaacaa attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc
     2641 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     2701 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     2761 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     2821 tcaccatgag tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag
     2881 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     2941 ttattcattc gtgattgcgc ctgagccaga cgaaatacgc gatcgctgtt aaaaggacaa
     3001 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     3061 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     3121 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     3181 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     3241 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     3301 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     3361 ttggaattta atcgcggcct cgagcaagac gtttcccgtt gaatatggct cataacaccc
     3421 cttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct
     3481 tgtgcaatgt aacatcagag attttgagac acaacgtggc tttcccccgc cgctctagaa
     3541 ctagtggatc caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     3601 ttgtttgtcg cattatagca gacgtccagg ttgggatacc tgaaacaaaa cccatcgtac
     3661 ggccaaggaa gtctc
//
