LOCUS       sequence-320858-        2478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Acceptor for golden gate assembly with BsaI overhangs GCTT-CCAT. 
            Generates plasmids for SP6-driven TNT SP6 Coupled Wheat Germ Extract
            System .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2478)
  AUTHORS   Dudley QM, Cai YM, Kallam K, Debreyne H, Carrasco Lopez JA, Patron 
            NJ
  TITLE     Biofoundry-assisted expression and characterization of plant 
            proteins.
  JOURNAL   Synth Biol (Oxf). 2021 Sep 11;6(1):ysab029. doi: 
            10.1093/synbio/ysab029. eCollection 2021.
  PUBMED    34693026
REFERENCE   2  (bases 1 to 2478)
  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: "Synth Biol 
            (Oxf)."; date: "2021-09-11"; volume: "6(1):ysab029. doi"; pages: " 
            10.1093/synbio/ysab029. eCollection 2021"
COMMENT     Sequence Label: pEPQD0KN0283
FEATURES             Location/Qualifiers
     source          1..2478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     172..194
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     186..203
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     187..203
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    204..260
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(273..289)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(273..289)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(286..308)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    297..313
                     /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        complement(join(321..327,328..345,346..351))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    366..387
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(498..516)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      512..559
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(577..1392)
                     /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="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1300..1319
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1384..1403
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1514..2102
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2003..2022
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2221..2239
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2221..2238
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(join(2432..2478,1..277))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     2437..2456
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca
       61 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
      121 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
      181 gacgttgtaa aacgacggcc agtgaattcg agctcggtac ccggggatcc tctagagtcg
      241 acctgcaggc atgcaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
      301 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
      361 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
      421 ggaaacctgt cgtgccagct aggtctcagc ttcggctgct aacaaagccc gaaaggaagc
      481 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
      541 ggtcttgagg ggttttttgc tgaaagccaa ttctgattag aaaaactcat cgagcatcaa
      601 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
      661 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
      721 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
      781 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
      841 cttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
      901 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
      961 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     1021 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     1081 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     1141 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     1201 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     1261 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     1321 atataaatca gcatccatgt tggaatttaa tcgcggcttc gagcaagacg tttcccgttg
     1381 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     1441 tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca caacgtgaga
     1501 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     1561 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     1621 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     1681 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     1741 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     1801 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     1861 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     1921 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     1981 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2041 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2101 aaaacgccag caacgcgatc ccgcgacgaa taagccagat gctacacaat taggcttgta
     2161 catactgtcg ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg
     2221 atttaggtga cactatagaa ctcacctatc tccccaacac ctaataacat tcaatcactc
     2281 tttccactaa ccacctatct acatcaccaa gatatcacta gttctcgagc tccatagaga
     2341 ccgtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     2401 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     2461 gagtgcacca tatgcggt
//
