LOCUS       sequence-291988-        5960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Chassis plasmid for building mutagenesis cassettes by Golden Gate 
            assembly to form a donor plasmid for recombineering by Gene 
            Doctoring.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5960)
  AUTHORS   Thomson NM, Zhang C, Trampari E, Pallen MJ
  TITLE     Creation of Golden Gate constructs for gene doctoring.
  JOURNAL   BMC Biotechnol. 2020 Oct 7;20(1):54. doi: 
            10.1186/s12896-020-00648-5.
  PUBMED    33028286
REFERENCE   2  (bases 1 to 5960)
  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: "BMC 
            Biotechnol."; date: "2020-10-7"; volume: "20(1):54. doi"; pages: " 
            10.1186/s12896-020-00648-5"
COMMENT     Sequence Label: pDOC-GG
FEATURES             Location/Qualifiers
     source          1..5960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        132..223
                     /gene="bla"
                     /label=AmpR promoter
     CDS             224..1033
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(291..310)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1195..1783
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1684..1703
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1884..1900
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1948..1969
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1984..1989,1990..2007,2008..2014)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2022..2038
                     /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)."
     primer_bind     2027..2049
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2046..2062
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2080..2096)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2418..2434)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2614..2631)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2772..2858
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2950..2977
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     oriT            3402..3511
                     /note="incP origin of transfer"
     promoter        3885..4330
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(4331..4417,4418..5752)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     primer_bind     5910..5932
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgtcaga attggttaat tggttgtaac
      121 actgacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
      181 ataaccctga taaatgcttc aataatattg aaaaaggaag aatatgagcc atattcaacg
      241 ggaaacgtcg aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
      301 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga
      361 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
      421 gatggtcaga ctaaactggc tgacggaatt tatgccactt ccgaccatca agcattttat
      481 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcgttcca
      541 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
      601 gcgccggttg cactcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
      661 cctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
      721 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
      781 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
      841 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
      901 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
      961 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     1021 tgagtttttc taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     1081 tcatttttaa tttaaaagga tctaggtgtt tgataatctc atgaccaaaa tcccttaacg
     1141 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     1201 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     1261 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     1321 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     1381 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     1441 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     1501 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     1561 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     1621 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     1681 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     1741 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     1801 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     1861 ccctgattct gtggataacc gtacaggaaa cagctatgac catgattacg ccaagctcta
     1921 gggataacag ggtaatctac tgagacctaa tgtgagttag ctcactcatt aggcacccca
     1981 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     2041 tcacacagga aacagctatg accatgatta cgtcggatta ctggccgtcg ttttacaacg
     2101 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     2161 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     2221 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2281 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     2341 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggcg gtctcaacga tagggataac
     2401 agggtaatga gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     2461 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     2521 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgagct
     2581 tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag
     2641 aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc
     2701 catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggcga gagtagggaa
     2761 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     2821 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     2881 ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc
     2941 aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actctttttg
     3001 tttatttttc taaatacatt caaatatgca tgcgcctgat gcggtatttt ctccttacgc
     3061 atatcgacat ccgccctcac cgccaggaac gcaaccgcag cctcatcacg ccggcgcttc
     3121 ttggccgcgc gggattcaac ccactcggcc agctcgtcgg tgtagctctt tggcatcgtc
     3181 tctcgcctgt cccctcagtt cagtaatttc ctgcatttgc ctgtttccag tcggtagata
     3241 ttccacaaaa cagcagggaa gcagcgcttt tccgctgcat aaccctgctt cggggtcatt
     3301 atagcgattt tttcggtata tccatccttt ttcgcacgat atacaggatt ttgccaaagg
     3361 gttcgtgtag actttccttg gtgtatccaa cggcgtcagc cgggcaggat aggtgaagta
     3421 ggcccacccg cgagcgggtg ttccttcttc actgtccctt attcgcacct ggcggtgctc
     3481 aacgggaatc ctgctctgcg aggctggccg gctaccgccg gcgtaacaga tgagggcaag
     3541 cggatggctg atgaaaccaa gccaaccagg aagggcagcc cacctatcaa ggtgtactgc
     3601 cttccagacg aacgaagagc gattgaggaa aaggcggcgg cggccggcat gagcctgtcg
     3661 gcctacctgc tggccgtcgg ccagggctac aaaatcacgg gcgtcgtgga ctatgagcac
     3721 gtccgcgagc tggcccgcat caatggcgac ctgggccgcc tgggcggcct gctgaaactc
     3781 tggctcaccg acgacccgcg cacggcgcgg ttcggtgatg ccacgatcct cgccctgctg
     3841 gcgaagatcg actctagcta gaggatcgat cctttttaac ccatcacata tacctgccgt
     3901 tcactattat ttagtgaaat gagatattat gatattttct gaattgtgat taaaaaggca
     3961 actttatgcc catgcaacag aaactataaa aaatacagag aatgaaaaga aacagataga
     4021 ttttttagtt ctttaggccc gtagtctgca aatcctttta tgattttcta tcaaacaaaa
     4081 gaggaaaata gaccagttgc aatccaaacg agagtctaat agaatgaggt cgaaaagtaa
     4141 atcgcgcggg tttgttactg ataaagcagg caagacctaa aatgtgtaaa gggcaaagtg
     4201 tatactttgg cgtcacccct tacatatttt aggtcttttt ttattgtgcg taactaactt
     4261 gccatcttca aacaggaggg ctggaagaag cagaccgcta acacagtaca taaaaaagga
     4321 gacatgaacg atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac
     4381 cgcactgctg gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc aaaagccata
     4441 taaggaaaca tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca
     4501 gcaaaaaaat gaaaaatatc aagttcctga gttcgattcg tccacaatta aaaatatctc
     4561 ttctgcaaaa ggcctggacg tttgggacag ctggccatta caaaacgctg acggcactgt
     4621 cgcaaactat cacggctacc acatcgtctt tgcattagcc ggagatccta aaaatgcgga
     4681 tgacacatcg atttacatgt tctatcaaaa agtcggcgaa acttctattg acagctggaa
     4741 aaacgctggc cgcgtcttta aagacagcga caaattcgat gcaaatgatt ctatcctaaa
     4801 agaccaaaca caagaatggt caggttcagc cacatttaca tctgacggaa aaatccgttt
     4861 attctacact gatttctccg gtaaacatta cggcaaacaa acactgacaa ctgcacaagt
     4921 taacgtatca gcatcagaca gctctttgaa catcaacggt gtagaggatt ataaatcaat
     4981 ctttgacggt gacggaaaaa cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta
     5041 cagctcaggc gacaaccata cgctgagaga tcctcactac gtagaagata aaggccacaa
     5101 atacttagta tttgaagcaa acactggaac tgaagatggc taccaaggcg aagaatcttt
     5161 atttaacaaa gcatactatg gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact
     5221 tctgcaaagc gataaaaaac gcacggctga gttagcaaac ggcgctctcg gtatgattga
     5281 gctaaacgat gattacacac tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt
     5341 aacagatgaa attgaacgcg cgaacgtctt taaaatgaac ggcaaatggt atctgttcac
     5401 tgactcccgc ggatcaaaaa tgacgattga cggcattacg tctaacgata tttacatgct
     5461 tggttatgtt tctaattctt taactggccc atacaagccg ctgaacaaaa ctggccttgt
     5521 gttaaaaatg gatcttgatc ctaacgatgt aacctttact tactcacact tcgctgtacc
     5581 tcaagcgaaa ggaaacaatg tcgtgattac aagctatatg acaaacagag gattctacgc
     5641 agacaaacaa tcaacgtttg cgcctagctt cctgctgaac atcaaaggca agaaaacatc
     5701 tgttgtcaaa gacagcatcc ttgaacaagg acaattaaca gttaacaaat aaaaacgcaa
     5761 aagaaaatgc cgattatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5821 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5881 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5941 gtcatcaccg aaacgcgcga
//
