LOCUS       sequence-435072-        3790 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 3790)
  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 3790)
  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-hPGK-HygroR-Z
FEATURES             Location/Qualifiers
     source          1..3790
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     35..57
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     49..66
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     50..66
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        146..656
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     575..593
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             667..1692
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             667..1692
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(1800..1816)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1800..1816)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1813..1835)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(1988..2576)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2068..2087)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2712..3527)
                     /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(2712..3527)
                     /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     3435..3454
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3519..3538
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     3648..3664
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3648..3664
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      3665..3711
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aataactgtg atccaccaca agcgccaggg ttttcccagt cacgacgttg taaaacgacg
       61 gccagtcatg cataatccgc acgcatctgg aataaggaag tgccattccg cctgacctga
      121 agacgtgtga cgtctccgca aacctggggt tggggttgcg ccttttccaa ggcagccctg
      181 ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc
      241 ctgggactcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc gccgctaccc
      301 ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt tccttgcggt
      361 tcgcggcgtg ccggacgtga caaacggaag ccgcacgcct cactagtacc ctcgcagacg
      421 gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct
      481 gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg
      541 gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg
      601 agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct ccccagaaca
      661 gccaccatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag
      721 ttcgacagcg tgtccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc
      781 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac
      841 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt
      901 gacattgggg aatttagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc
      961 acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc
     1021 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg
     1081 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat
     1141 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc
     1201 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat
     1261 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc
     1321 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg
     1381 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga
     1441 tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg
     1501 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga
     1561 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc
     1621 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg
     1681 gcaaaggaat agggctctaa ggttaagtcg ccctcggtat cagatgagac gcgtatagtc
     1741 ttcaggctag gtggaggctc agtgatgata agtctgcgat ggtggatgca tgtgtcatgg
     1801 tcatagctgt ttcctgtgtg aaattgttat ccgctcagag ggcacaatcc tattccgcgc
     1861 tatccgacaa tctccaagac attaggtgga gttcagttcg gcgtatggca tatgtcgctg
     1921 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     1981 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2041 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2101 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2161 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2221 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2281 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2341 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2401 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     2461 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2521 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     2581 atcctttgat cttttctacg gggtctgacg ctctattcaa caaagccgcc gtcccgtcaa
     2641 gtcagcgtaa atgggtaggg ggcttcaaat cgtccgctct gccagtgtta caaccaatta
     2701 acaaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     2761 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     2821 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     2881 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     2941 tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt
     3001 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     3061 attcgtgatt gcgcctgagc cagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     3121 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     3181 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     3241 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     3301 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     3361 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     3421 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     3481 tttaatcgcg gcctcgagca agacgtttcc cgttgaatat ggctcataac accccttgta
     3541 ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca
     3601 atgtaacatc agagattttg agacacaacg tggctttccc ccgccgctct agaactagtg
     3661 gatccaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     3721 gtcgcattat agcagacgtc caggttggga tacctgaaac aaaacccatc gtacggccaa
     3781 ggaagtctcc
//
