LOCUS       sequence-308794-        5983 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 5983)
  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 5983)
  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..5983
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(6..24)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        128..219
                     /gene="bla"
                     /label=AmpR promoter
     CDS             220..1029
                     /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(287..306)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1191..1779
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1680..1699
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1880..1896
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1944..1965
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1980..1985,1986..2003,2004..2010)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2018..2034
                     /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     2023..2045
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2042..2058
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2054..2326
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITSDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDAA"
     primer_bind     complement(2077..2093)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2415..2431)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2611..2628)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2769..2855
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2947..2974
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     oriT            3421..3530
                     /note="incP origin of transfer"
     promoter        3904..4349
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(4350..4436,4437..5771)
                     /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     5929..5951
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
       61 gacgtcaggt ggcacttttc ggggaaatgt gtcagaattg gttaattggt tgtaacactg
      121 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
      181 ccctgataaa tgcttcaata atattgaaaa aggaagaata tgagccatat tcaacgggaa
      241 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
      301 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
      361 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
      421 gtcagactaa actggctgac ggaatttatg ccacttccga ccatcaagca ttttatccgt
      481 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc gttccaggta
      541 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
      601 cggttgcact cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgcctc
      661 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
      721 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
      781 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
      841 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
      901 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
      961 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     1021 tttttctaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     1081 ttttaattta aaaggatcta ggtgtttgat aatctcatga ccaaaatccc ttaacgtgag
     1141 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     1201 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     1261 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     1321 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     1381 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     1441 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     1501 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1561 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1621 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1681 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     1741 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     1801 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     1861 gattctgtgg ataaccgtac aggaaacagc tatgaccatg attacgccaa gctctaggga
     1921 taacagggta atctactgag acctaatgtg agttagctca ctcattaggc accccaggct
     1981 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     2041 acaggaaaca gctatgacca tgattacgtc ggattcactg gccgtcgttt tacaacgtcg
     2101 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     2161 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     2221 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2281 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     2341 acacccgcca acacccgctg acgcgccctg acgggcggtc tcaacgatag ggataacagg
     2401 gtaatgagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     2461 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     2521 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgagcttgg
     2581 ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag
     2641 cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat
     2701 gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggcgagag tagggaactg
     2761 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     2821 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     2881 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa
     2941 ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttttgttta
     3001 tttttctaaa tacattcaaa tatgcatgcg cctgatgcgg tattttctcc ttacgcatct
     3061 gtgcggtatt tcacaccgca tatcgacatc cgccctcacc gccaggaacg caaccgcagc
     3121 ctcatcacgc cggcgcttct tggccgcgcg ggattcaacc cactcggcca gctcgtcggt
     3181 gtagctcttt ggcatcgtct ctcgcctgtc ccctcagttc agtaatttcc tgcatttgcc
     3241 tgtttccagt cggtagatat tccacaaaac agcagggaag cagcgctttt ccgctgcata
     3301 accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt tcgcacgata
     3361 tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc
     3421 gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca ctgtccctta
     3481 ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgg ctaccgccgg
     3541 cgtaacagat gagggcaagc ggatggctga tgaaaccaag ccaaccagga agggcagccc
     3601 acctatcaag gtgtactgcc ttccagacga acgaagagcg attgaggaaa aggcggcggc
     3661 ggccggcatg agcctgtcgg cctacctgct ggccgtcggc cagggctaca aaatcacggg
     3721 cgtcgtggac tatgagcacg tccgcgagct ggcccgcatc aatggcgacc tgggccgcct
     3781 gggcggcctg ctgaaactct ggctcaccga cgacccgcgc acggcgcggt tcggtgatgc
     3841 cacgatcctc gccctgctgg cgaagatcga ctctagctag aggatcgatc ctttttaacc
     3901 catcacatat acctgccgtt cactattatt tagtgaaatg agatattatg atattttctg
     3961 aattgtgatt aaaaaggcaa ctttatgccc atgcaacaga aactataaaa aatacagaga
     4021 atgaaaagaa acagatagat tttttagttc tttaggcccg tagtctgcaa atccttttat
     4081 gattttctat caaacaaaag aggaaaatag accagttgca atccaaacga gagtctaata
     4141 gaatgaggtc gaaaagtaaa tcgcgcgggt ttgttactga taaagcaggc aagacctaaa
     4201 atgtgtaaag ggcaaagtgt atactttggc gtcacccctt acatatttta ggtctttttt
     4261 tattgtgcgt aactaacttg ccatcttcaa acaggagggc tggaagaagc agaccgctaa
     4321 cacagtacat aaaaaaggag acatgaacga tgaacatcaa aaagtttgca aaacaagcaa
     4381 cagtattaac ctttactacc gcactgctgg caggaggcgc aactcaagcg tttgcgaaag
     4441 aaacgaacca aaagccatat aaggaaacat acggcatttc ccatattaca cgccatgata
     4501 tgctgcaaat ccctgaacag caaaaaaatg aaaaatatca agttcctgag ttcgattcgt
     4561 ccacaattaa aaatatctct tctgcaaaag gcctggacgt ttgggacagc tggccattac
     4621 aaaacgctga cggcactgtc gcaaactatc acggctacca catcgtcttt gcattagccg
     4681 gagatcctaa aaatgcggat gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa
     4741 cttctattga cagctggaaa aacgctggcc gcgtctttaa agacagcgac aaattcgatg
     4801 caaatgattc tatcctaaaa gaccaaacac aagaatggtc aggttcagcc acatttacat
     4861 ctgacggaaa aatccgttta ttctacactg atttctccgg taaacattac ggcaaacaaa
     4921 cactgacaac tgcacaagtt aacgtatcag catcagacag ctctttgaac atcaacggtg
     4981 tagaggatta taaatcaatc tttgacggtg acggaaaaac gtatcaaaat gtacagcagt
     5041 tcatcgatga aggcaactac agctcaggcg acaaccatac gctgagagat cctcactacg
     5101 tagaagataa aggccacaaa tacttagtat ttgaagcaaa cactggaact gaagatggct
     5161 accaaggcga agaatcttta tttaacaaag catactatgg caaaagcaca tcattcttcc
     5221 gtcaagaaag tcaaaaactt ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg
     5281 gcgctctcgg tatgattgag ctaaacgatg attacacact gaaaaaagtg atgaaaccgc
     5341 tgattgcatc taacacagta acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg
     5401 gcaaatggta tctgttcact gactcccgcg gatcaaaaat gacgattgac ggcattacgt
     5461 ctaacgatat ttacatgctt ggttatgttt ctaattcttt aactggccca tacaagccgc
     5521 tgaacaaaac tggccttgtg ttaaaaatgg atcttgatcc taacgatgta acctttactt
     5581 actcacactt cgctgtacct caagcgaaag gaaacaatgt cgtgattaca agctatatga
     5641 caaacagagg attctacgca gacaaacaat caacgtttgc gcctagcttc ctgctgaaca
     5701 tcaaaggcaa gaaaacatct gttgtcaaag acagcatcct tgaacaagga caattaacag
     5761 ttaacaaata aaaacgcaaa agaaaatgcc gattatggtg cactctcagt acaatctgct
     5821 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     5881 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     5941 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acg
//
