LOCUS       sequence-435070-        3278 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 3278)
  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 3278)
  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-BlastR-SV40 polyA-Z
FEATURES             Location/Qualifiers
     source          1..3278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(84..100)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(84..100)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(97..119)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(272..860)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(352..371)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(996..1811)
                     /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(996..1811)
                     /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     1719..1738
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1803..1822
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     1932..1948
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1932..1948
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      1949..1995
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2109..2131
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2123..2140
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2124..2140
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2227..2726
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2262..2285)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2659..2678
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2739..3137
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(3180..3199)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3234..3253
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 gtatcagatg agacgcgtat agtcttcagg ctaggtggag gctcagtgat gataagtctg
       61 cgatggtgga tgcatgtgtc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
      121 agagggcaca atcctattcc gcgctatccg acaatctcca agacattagg tggagttcag
      181 ttcggcgtat ggcatatgtc gctggaaaga acatgtgagc aaaaggccag caaaaggcca
      241 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
      301 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
      361 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
      421 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
      481 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
      541 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
      601 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
      661 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
      721 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
      781 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
      841 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctctat
      901 tcaacaaagc cgccgtcccg tcaagtcagc gtaaatgggt agggggcttc aaatcgtccg
      961 ctctgccagt gttacaacca attaacaaat tctgattaga aaaactcatc gagcatcaaa
     1021 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     1081 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     1141 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     1201 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc
     1261 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     1321 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagccagacg aaatacgcga
     1381 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     1441 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     1501 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     1561 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     1621 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     1681 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     1741 tataaatcag catccatgtt ggaatttaat cgcggcctcg agcaagacgt ttcccgttga
     1801 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     1861 gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt
     1921 tcccccgccg ctctagaact agtggatcca aataaaacga aaggctcagt cgaaagactg
     1981 ggcctttcgt tttatctgtt gtttgtcgca ttatagcaga cgtccaggtt gggatacctg
     2041 aaacaaaacc catcgtacgg ccaaggaagt ctccaataac tgtgatccac cacaagcgcc
     2101 agggttttcc cagtcacgac gttgtaaaac gacggccagt catgcataat ccgcacgcat
     2161 ctggaataag gaagtgccat tccgcctgac ctgaagacgt gtgacgtctc cgcaaacctt
     2221 tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc
     2281 cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg
     2341 gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc
     2401 tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag
     2461 tagcacgcct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag
     2521 gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg
     2581 gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa
     2641 ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct
     2701 cctcttcctc atctccgggc ctttcgacct aacagaccat ggccaagcct ttgtctcaag
     2761 aagaatccac cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag
     2821 actacagcgt cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg
     2881 tatatcattt tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg
     2941 cggcagctgg caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga
     3001 gcccctgcgg acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag
     3061 tgaaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt
     3121 atgtgtggga gggctaaggc tccacttgtt tattgcagct tataatggtt acaaataaag
     3181 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     3241 gtccaaactc atcaatgtat cttatcatgt ctggctct
//
