LOCUS       sequence-434965-        3654 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 3654)
  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 3654)
  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-W-A-ccdB-G-X
FEATURES             Location/Qualifiers
     source          1..3654
                     /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"
     CDS             complement(159..464)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(261..280)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(806..1465)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1380..1399
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1519..1525,1526..1543,1544..1549))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(1664..1680)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1664..1680)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1677..1699)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(1852..2440)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1932..1951)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2576..3391)
                     /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(2576..3391)
                     /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     3299..3318
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3383..3402
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     3512..3528
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3512..3528
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      3529..3575
                     /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 aaccttgaga ccgtcgactt atattcccca gaacatcagg
      181 ttaatggcgt ttttgatgtc attttcgcgg tggctgagat cagccacttc ttccccgata
      241 acggagactg gcacactggc catatcggtg gtcatcatgc gccagctttc atccccgata
      301 tgcaccaccg ggtaaagttc acgggagact ttatctgaca gcagacgtgc actggccagg
      361 gggatcacca tccgtcgccc gggcgtgtca ataatatcac tctgtacatc cacaaacaga
      421 cgataacggc tctctctttt ataggtgtaa accttaaact gcatttcacc agtccctgtt
      481 ctcgtcagca aaagagccgt tcatttcaat aaaccgggcg acctcagcca tcccttcctg
      541 attttccgct ttccagcgtt cggcacgcag acgacgggct tcattctgca tggttgtgct
      601 taccagaccg gagatattga catcatatat gccttgagca actgatagct gtcgctgtca
      661 actgtcactg taatacgctg cttcatagca cacctctttt tgacatactt cgggtataca
      721 tatcagtata tattcttata ccgcaaaaat cagcgcgcaa atacgcatac tgttatctgg
      781 cttttagtaa gccggatcca cgcgtttacg ccccgccctg ccactcatcg cagtactgtt
      841 gtaattcatt aagcattctg ccgacatgga agccatcaca gacggcatga tgaacctgaa
      901 tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg
      961 gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg
     1021 gattggctga cacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt
     1081 caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt
     1141 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt
     1201 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag
     1261 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct
     1321 ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag
     1381 caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg
     1441 tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat ctcgccggat
     1501 cctaactcaa aatccacaca ttatacgagc cggaagcata aagtgtaaag cctggggtgc
     1561 ctaatgcggc cgcggtctca gtatcagatg agacgcgtat agtcttcagg ctaggtggag
     1621 gctcagtgat gataagtctg cgatggtgga tgcatgtgtc atggtcatag ctgtttcctg
     1681 tgtgaaattg ttatccgctc agagggcaca atcctattcc gcgctatccg acaatctcca
     1741 agacattagg tggagttcag ttcggcgtat ggcatatgtc gctggaaaga acatgtgagc
     1801 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     1861 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     1921 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     1981 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2041 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagccggg
     2101 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2161 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2221 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     2281 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2341 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     2401 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     2461 tacggggtct gacgctctat tcaacaaagc cgccgtcccg tcaagtcagc gtaaatgggt
     2521 agggggcttc aaatcgtccg ctctgccagt gttacaacca attaacaaat tctgattaga
     2581 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     2641 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     2701 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     2761 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     2821 ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat
     2881 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     2941 gagccagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     3001 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     3061 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     3121 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     3181 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     3241 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     3301 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg
     3361 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     3421 cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat
     3481 tttgagacac aacgtggctt tcccccgccg ctctagaact agtggatcca aataaaacga
     3541 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcgca ttatagcaga
     3601 cgtccaggtt gggatacctg aaacaaaacc catcgtacgg ccaaggaagt ctcc
//
