LOCUS       sequence-434966-        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-X-A-ccdB-G-Z
FEATURES             Location/Qualifiers
     source          1..3654
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     36..58
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     50..67
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     51..67
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(160..465)
                     /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(262..281)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(807..1466)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1381..1400
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1520..1526,1527..1544,1545..1550))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(1665..1681)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1665..1681)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1678..1700)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(1853..2441)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1933..1952)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2577..3392)
                     /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(2577..3392)
                     /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     3300..3319
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3384..3403
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     3513..3529
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3513..3529
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      3530..3576
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 caataactgt gatccaccac aagcgccaga gttttcccag tcacgacgtt gtaaaacgac
       61 ggccagtcat gcataatccg cacgcatctg gaataaggaa gtgccattcc gcctgacctg
      121 aagacgtgtg acgtctcctg ccaccttgag accgtcgact tatattcccc agaacatcag
      181 gttaatggcg tttttgatgt cattttcgcg gtggctgaga tcagccactt cttccccgat
      241 aacggagact ggcacactgg ccatatcggt ggtcatcatg cgccagcttt catccccgat
      301 atgcaccacc gggtaaagtt cacgggagac tttatctgac agcagacgtg cactggccag
      361 ggggatcacc atccgtcgcc cgggcgtgtc aataatatca ctctgtacat ccacaaacag
      421 acgataacgg ctctctcttt tataggtgta aaccttaaac tgcatttcac cagtccctgt
      481 tctcgtcagc aaaagagccg ttcatttcaa taaaccgggc gacctcagcc atcccttcct
      541 gattttccgc tttccagcgt tcggcacgca gacgacgggc ttcattctgc atggttgtgc
      601 ttaccagacc ggagatattg acatcatata tgccttgagc aactgatagc tgtcgctgtc
      661 aactgtcact gtaatacgct gcttcatagc acacctcttt ttgacatact tcgggtatac
      721 atatcagtat atattcttat accgcaaaaa tcagcgcgca aatacgcata ctgttatctg
      781 gcttttagta agccggatcc acgcgtttac gccccgccct gccactcatc gcagtactgt
      841 tgtaattcat taagcattct gccgacatgg aagccatcac agacggcatg atgaacctga
      901 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg
      961 ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag
     1021 ggattggctg acacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt
     1081 tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg
     1141 tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg
     1201 tgaacactat cccatatcac cagctcaccg tctttcattg ccatacggaa ttccggatga
     1261 gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc
     1321 tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga
     1381 gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg
     1441 gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgccgga
     1501 tcctaactca aaatccacac attatacgag ccggaagcat aaagtgtaaa gcctggggtg
     1561 cctaatgcgg ccgcggtctc agtatgcaat gagacgcgta tagtcttcag gctaggtgga
     1621 ggctcagtga tgataagtct gcgatggtgg atgcatgtgt catggtcata gctgtttcct
     1681 gtgtgaaatt gttatccgct cagagggcac aatcctattc cgcgctatcc gacaatctcc
     1741 aagacattag gtggagttca gttcggcgta tggcatatgt cgctggaaag aacatgtgag
     1801 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     1861 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     1921 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1981 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2041 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2101 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2161 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2221 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2281 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2341 aaagagttgg tagctcttga tccggcaaaa aaaccaccgc tggtagcggt ggtttttttg
     2401 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2461 ctacggggtc tgacgctcta ttcaacaaag ccgccgtccc gtcaagtcag cgtaaatggg
     2521 tagggggctt caaatcgtcc gctctgccag tgttacaacc aattaacaaa ttctgattag
     2581 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     2641 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     2701 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     2761 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     2821 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca
     2881 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     2941 tgagccagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     3001 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     3061 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     3121 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     3181 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     3241 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     3301 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc
     3361 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     3421 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga
     3481 ttttgagaca caacgtggct ttcccccgcc gctctagaac tagtggatcc aaataaaacg
     3541 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgc attatagcag
     3601 acgtccaggt tgggatacct gaaacaaaac ccatcgtacg gccaaggaag tctc
//
