LOCUS       sequence-435065-        3698 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 3698)
  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 3698)
  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: pGGE-V5_TurboID-F
FEATURES             Location/Qualifiers
     source          1..3698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             14..55
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             62..1018
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG
                     ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI
                     TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK
                     EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     primer_bind     complement(1044..1063)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1045..1063)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1073..1095)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1289..1308)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1408..1430
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1468..1486)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1554..1658
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1659..1727,1728..2519)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2690..3278
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3179..3198
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3432..3449
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3566..3587
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3602..3607,3608..3625,3626..3632)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3640..3656
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        3670..3688
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3670..3687
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 ggtctcactg cagggcaagc ccatccccaa ccccctgctg ggcctggaca gcaccgctag
       61 caaagacaat actgtgcctc tgaagctgat cgctctcctg gctaatggcg agttccatag
      121 tggcgaacag ctgggagaaa ccctgggcat gtccagggcc gctatcaaca agcacattca
      181 gactctgcgc gactggggcg tggacgtgtt caccgtgccc ggaaagggct actctctgcc
      241 cgagcctatc ccgctgctga acgctaaaca gattctggga cagctggacg gcgggagcgt
      301 ggcagtcctg cctgtggtcg actccaccaa tcagtacctg ctggatcgaa tcggcgagct
      361 gaagagtggg gatgcttgca ttgcagaata tcagcaggca gggagaggaa gcagagggag
      421 gaaatggttc tctccttttg gagctaacct gtacctgagt atgttttggc gcctgaagcg
      481 gggaccagca gcaatcggcc tgggcccggt catcggaatt gtcatggcag aagcgctgcg
      541 aaagctggga gcagacaagg tgcgagtcaa atggcccaat gacctgtatc tgcaggatag
      601 aaagctggca ggcatcctgg tggagctggc cggaataaca ggcgatgctg cacagatcgt
      661 cattggcgcc gggattaacg tggctatgag gcgcgtggag gaaagcgtgg tcaatcaggg
      721 ctggatcaca ctgcaggaag cagggattaa cctggacagg aatactctgg ccgctacgct
      781 gatccgagag ctgcgggcag ccctggaact gttcgagcag gaaggcctgg ctccatatct
      841 gccacggtgg gagaagctgg ataacttcat caatagaccc gtgaagctga tcattgggga
      901 caaagagatt ttcgggatta gccgggggat tgataaacag ggagccctgc tgctggaaca
      961 ggacggagtt atcaaaccct ggatgggcgg agaaatcagt ctgcggtctg ccgaaaagta
     1021 atgactatga gaccgaattc tcgccctata gtgagtcgta ttacctggcc gtcgttttac
     1081 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     1141 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     1201 gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     1261 tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     1321 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     1381 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     1441 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     1501 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     1561 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1621 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     1681 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     1741 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     1801 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1861 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1921 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1981 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     2041 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     2101 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     2161 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     2221 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     2281 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     2341 ttattgctga taaatctgga gccggtgagc gtggctctcg aggtatcatt gcagcactgg
     2401 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     2461 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     2521 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     2581 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     2641 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2701 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2761 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2821 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2881 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2941 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3001 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3061 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3121 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3181 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3241 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3301 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3361 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3421 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     3481 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     3541 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     3601 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     3661 acacgagcta tttaggtgac actatagaag tgaagctt
//
