LOCUS       sequence-435074-        3558 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 3558)
  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 3558)
  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-NeoR-Z
FEATURES             Location/Qualifiers
     source          1..3558
                     /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             666..1460
                     /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             666..1460
                     /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(720..739)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1330..1349
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1568..1584)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1568..1584)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1581..1603)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(1756..2344)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1836..1855)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2480..3295)
                     /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(2480..3295)
                     /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     3203..3222
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3287..3306
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     3416..3432
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3416..3432
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      3433..3479
                     /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 ccaccatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
      721 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
      781 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
      841 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
      901 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
      961 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     1021 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     1081 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     1141 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     1201 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     1261 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     1321 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     1381 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     1441 ttcttgacga gttcttctga ggctctaagg ttaagtcgcc ctcggtatca gatgagacgc
     1501 gtatagtctt caggctaggt ggaggctcag tgatgataag tctgcgatgg tggatgcatg
     1561 tgtcatggtc atagctgttt cctgtgtgaa attgttatcc gctcagaggg cacaatccta
     1621 ttccgcgcta tccgacaatc tccaagacat taggtggagt tcagttcggc gtatggcata
     1681 tgtcgctgga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     1741 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     1801 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     1861 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     1921 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     1981 gtaggtcgtt cgctccaagc cgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2041 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2101 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2161 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     2221 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2281 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     2341 tcaagaagat cctttgatct tttctacggg gtctgacgct ctattcaaca aagccgccgt
     2401 cccgtcaagt cagcgtaaat gggtaggggg cttcaaatcg tccgctctgc cagtgttaca
     2461 accaattaac aaattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat
     2521 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     2581 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     2641 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     2701 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc
     2761 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     2821 cgttattcat tcgtgattgc gcctgagcca gacgaaatac gcgatcgctg ttaaaaggac
     2881 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     2941 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     3001 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     3061 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     3121 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     3181 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     3241 tgttggaatt taatcgcggc ctcgagcaag acgtttcccg ttgaatatgg ctcataacac
     3301 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat
     3361 cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttccccc gccgctctag
     3421 aactagtgga tccaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     3481 tgttgtttgt cgcattatag cagacgtcca ggttgggata cctgaaacaa aacccatcgt
     3541 acggccaagg aagtctcc
//
