LOCUS       sequence-435060-        2701 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 2701)
  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 2701)
  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: pGGB-Kozak-ATG-C
FEATURES             Location/Qualifiers
     source          1..2701
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     72..91
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     325..342
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    459..480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(495..500,501..518,519..525)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    533..549
                     /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        563..581
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     563..580
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      609..618
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(638..657)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(639..657)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(667..689)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(883..902)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1002..1024
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1062..1080)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1148..1252
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1253..1321,1322..2113)
                     /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"
     primer_bind     complement(1471..1490)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(2284..2701,1..171)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
       61 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      121 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      181 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      241 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      301 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      361 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      421 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
      481 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
      541 ggataacaat ttcacacgag ctatttaggt gacactatag aagtgaagct tggtctcaaa
      601 catccgccgc caccatgggc ttgagaccga attctcgccc tatagtgagt cgtattacct
      661 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
      721 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
      781 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
      841 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
      901 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
      961 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     1021 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     1081 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     1141 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1201 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     1261 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     1321 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     1381 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     1441 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     1501 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     1561 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     1621 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     1681 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     1741 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     1801 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     1861 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     1921 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggct ctcgaggtat
     1981 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     2041 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     2101 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     2161 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     2221 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2281 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2341 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2401 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     2461 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2521 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2581 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2641 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2701 a
//
