LOCUS       sequence-435063-        2751 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 2751)
  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 2751)
  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: pGGD-T2A-E
FEATURES             Location/Qualifiers
     source          1..2751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(98..166,167..958)
                     /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(316..335)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1129..1717
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1618..1637
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1871..1888
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2005..2026
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2041..2046,2047..2064,2065..2071)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2079..2095
                     /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        2109..2127
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2109..2126
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             2160..2213
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(2234..2253)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2235..2253)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2263..2285)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2479..2498)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2598..2620
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2658..2676)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2744..2751,1..97)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
       61 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
      121 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
      181 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
      241 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
      301 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
      361 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
      421 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
      481 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
      541 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
      601 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
      661 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
      721 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
      781 tattgctgat aaatctggag ccggtgagcg tggctctcga ggtatcattg cagcactggg
      841 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
      901 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
      961 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     1021 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     1081 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     1141 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     1201 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     1261 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     1321 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     1381 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     1441 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     1501 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     1561 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     1621 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     1681 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     1741 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     1801 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     1861 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     1921 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     1981 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     2041 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     2101 cacgagctat ttaggtgaca ctatagaagt gaagcttggt ctcatcaggc ggctccggag
     2161 agggcagggg aagtcttcta acatgcgggg acgtggagga aaatcccggc cccctgctga
     2221 gaccgaattc tcgccctata gtgagtcgta ttacctggcc gtcgttttac aacgtcgtga
     2281 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     2341 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     2401 tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     2461 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     2521 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     2581 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     2641 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     2701 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa c
//
