LOCUS       sequence-435092-        3481 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 3481)
  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 3481)
  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-PuroR-SV40 polyA-Z
FEATURES             Location/Qualifiers
     source          1..3481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(54..100)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(101..117)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(101..117)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(227..246)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             238..1053
                     /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             238..1053
                     /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     complement(311..330)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1189..1777
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1678..1697
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1930..1952
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1949..1965
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1949..1965
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2074..2093)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2128..2147
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2190..2789)
                     /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(2273..2293)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2770..2789
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(2804..3303)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2852..2871)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     3245..3268
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     complement(3390..3407)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3390..3406)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3399..3421)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttttgtttca ggtatcccaa cctggacgtc tgctataatg cgacaaacaa cagataaaac
       61 gaaaggccca gtctttcgac tgagcctttc gttttatttg gatccactag ttctagagcg
      121 gcgggggaaa gccacgttgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat
      181 atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg
      241 agccatattc aacgggaaac gtcttgctcg aggccgcgat taaattccaa catggatgct
      301 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
      361 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
      421 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
      481 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
      541 cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
      601 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
      661 aacagcgatc gcgtatttcg tctggctcag gcgcaatcac gaatgaataa cggtttggtt
      721 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
      781 atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
      841 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
      901 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
      961 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     1021 cagtttcatt tgatgctcga tgagtttttc taatcagaat ttgttaattg gttgtaacac
     1081 tggcagagcg gacgatttga agccccctac ccatttacgc tgacttgacg ggacggcggc
     1141 tttgttgaat agagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     1201 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     1261 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     1321 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     1381 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     1441 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     1501 gggctgaacg gggggttcgt gcacacagcc cggcttggag cgaacgacct acaccgaact
     1561 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     1621 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     1681 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     1741 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     1801 acggttcctg gccttttgct ggccttttgc tcacatgttc tttccagcga catatgccat
     1861 acgccgaact gaactccacc taatgtcttg gagattgtcg gatagcgcgg aataggattg
     1921 tgccctctga gcggataaca atttcacaca ggaaacagct atgaccatga cacatgcatc
     1981 caccatcgca gacttatcat cactgagcct ccacctagcc tgaagactat acgcgtctca
     2041 tctgatacag agccagacat gataagatac attgatgagt ttggacaaac cacaactaga
     2101 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     2161 attataagct gcaataaaca agtggagcct caggcaccgg gcttgcgggt catgcaccag
     2221 gtgcgcggtc cttcgggcac ctcgacgtcg gcggtgacgg tgaagccgag ccgctcgtag
     2281 aaggggaggt tgcggggcgc ggaggtttcc aggaaggcgg gcaccccggc gcgctcggcc
     2341 gcctccactc cggggagcac gacggcgctg cccagaccct tgccctggtg gtcgggcgaa
     2401 acgccgacgg tggccaggaa ccacgcgggc tccttgggcc ggtgcggcgc caggaggcct
     2461 tccatctgtt gctgcgcggc cagccgggaa ccgctcaact cggccatgcg cgggccgatc
     2521 tcggcgaaca ccgcccccgc ttcgacgctc tccggcgtgg tccagaccgc caccgcggcg
     2581 ccgtcgtccg cgacccacac cttgccgatg tcgagcccga cgcgcgtgag gaagagttct
     2641 tgcagctcgg tgacccgctc gatgtggcgg tccggatcga cggtgtggcg cgtggcgggg
     2701 tagtcggcga acgcggcggc gagggtgcgt acggccctgg ggacgtcgtc gcgggtggcg
     2761 aggcgcaccg tgggcttgta ctcggtcatg gtggctgtta ggtcgaaagg cccggagatg
     2821 aggaagagga gaacagcgcg gcagacgtgc gcttttgaag cgtgcagaat gccgggcctc
     2881 cggaggacct tcgggcgccc gccccgcccc tgagcccgcc cctgagcccg cccccggacc
     2941 caccccttcc cagcctctga gcccagaaag cgaaggagca aagctgctat tggccgctgc
     3001 cccaaaggcc tacccgcttc cattgctcag cggtgctgtc catctgcacg agactagtga
     3061 ggcgtgctac ttccatttgt cacgtcctgc acgacgcgag ctgcggggcg ggggggaact
     3121 tcctgactag gggaggagta gaaggtggcg cgaaggggcc accaaagaac ggagccggtt
     3181 ggcgcctacc ggtggatgtg gaatgtgtgc gaggccagag gccacttgtg tagcgccaag
     3241 tgcccagcgg ggctgctaaa gcgcatgctc cagactgcct tgggaaaagc gcctccccta
     3301 cccggtagaa aggtttgcgg agacgtcaca cgtcttcagg tcaggcggaa tggcacttcc
     3361 ttattccaga tgcgtgcgga ttatgcatga ctggccgtcg ttttacaacg tcgtgactgg
     3421 gaaaaccctg gcgcttgtgg tggatcacag ttattggaga cttccttggc cgtacgatgg
     3481 g
//
