LOCUS       sequence-435071-        3364 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 3364)
  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 3364)
  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-PuroR-Z
FEATURES             Location/Qualifiers
     source          1..3364
                     /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..1266
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(667..686)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1163..1183
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1374..1390)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1374..1390)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1387..1409)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(1562..2150)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1642..1661)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2286..3101)
                     /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(2286..3101)
                     /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     3009..3028
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3093..3112
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     3222..3238
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3222..3238
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      3239..3285
                     /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 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
      721 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
      781 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
      841 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
      901 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
      961 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     1021 aaggagcccg cgtggttcct ggccaccgtc ggcgtttcgc ccgaccacca gggcaagggt
     1081 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     1141 ttcctggaaa cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     1201 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     1261 gcctgaggct ctaaggttaa gtcgccctcg gtatcagatg agacgcgtat agtcttcagg
     1321 ctaggtggag gctcagtgat gataagtctg cgatggtgga tgcatgtgtc atggtcatag
     1381 ctgtttcctg tgtgaaattg ttatccgctc agagggcaca atcctattcc gcgctatccg
     1441 acaatctcca agacattagg tggagttcag ttcggcgtat ggcatatgtc gctggaaaga
     1501 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     1561 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     1621 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     1681 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     1741 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     1801 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     1861 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     1921 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     1981 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     2041 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     2101 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     2161 tgatcttttc tacggggtct gacgctctat tcaacaaagc cgccgtcccg tcaagtcagc
     2221 gtaaatgggt agggggcttc aaatcgtccg ctctgccagt gttacaacca attaacaaat
     2281 tctgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2341 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2401 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2461 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2521 acgactgaat ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca
     2581 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2641 gattgcgcct gagccagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2701 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2761 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     2821 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2881 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2941 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     3001 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     3061 cgcggcctcg agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     3121 tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa
     3181 catcagagat tttgagacac aacgtggctt tcccccgccg ctctagaact agtggatcca
     3241 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcgca
     3301 ttatagcaga cgtccaggtt gggatacctg aaacaaaacc catcgtacgg ccaaggaagt
     3361 ctcc
//
