LOCUS       sequence-320882-        3129 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 3129)
  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 3129)
  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: pEPQD0KN0285
FEATURES             Location/Qualifiers
     source          1..3129
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     81..100
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      211..799
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     700..719
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        918..936
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     918..935
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(1129..1452)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     1134..1153
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1347..1369
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1361..1378
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1362..1378
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1379..1435
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1448..1464)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1472..1488
                     /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(1496..1502,1503..1520,1521..1526))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1541..1562
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2284..2300)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2324..2342)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2338..2385
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(join(2403..3129,1..89))
                     /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     join(3126..3129,1..16)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcttcgag
       61 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
      121 gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt
      181 tgagacacaa cgtgagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
      241 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
      301 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
      361 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
      421 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
      481 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
      541 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
      601 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
      661 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
      721 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
      781 ggggcggagc ctatggaaaa acgccagcaa cgcgatcccg cgacgaataa gccagatgct
      841 acacaattag gcttgtacat actgtcgtta gaacgcggct acaattaata cataacctta
      901 tgtatcatac acatacgatt taggtgacac tatagaactc acctatctcc ccaacaccta
      961 ataacattca atcactcttt ccactaacca cctatctaca tcaccaagat atcactagtt
     1021 ctcgagctcc atagagaccg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     1081 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     1141 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     1201 gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg
     1261 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     1321 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
     1381 attcgagctc ggtacccggg gatcctctag agtcgacctg caggcatgca agcttggcgt
     1441 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     1501 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     1561 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctaggtc
     1621 tcagcttcgg gttataatta cctcaggtcg acgtcccatg gttttgtata gaatttacgg
     1681 ctagcgccgg atgcgacgcc ggtcgcgtct tatccggcct tcctatatca ggcggtgttt
     1741 aagacgccgc cgcttcgccc aaatccttat gccggttcga cgactggaca aaatactgtt
     1801 tatcttccca gcgcaggcag gttaatgtac caccccagca gcagccggta tccagcgcgt
     1861 atataccttc cggcgtacct ttgccctcca gcgatgccca gtgaccaaag gcgatgctgt
     1921 attcttcagc gacagggcca ggaatcgcaa accacggttt cagtggggca ggggcctctt
     1981 ccggcgattc ttactagcta gtatgcatag gtgctgaaat ataaagtttg tgtttctaaa
     2041 acacacgtgg tacgtacgat aacgtacagt gtttttccct ccacttaaat cgaagggtag
     2101 tgtcttggag cgcgcggagt aaacatatat ggttcatata tgtccgtagg cacgtaaaaa
     2161 aagcgaggga ttcgaattcc cccggaaccc ccggttgggg cccacgcctc gatcgagcaa
     2221 aaaaaaaaaa aangaaaaaa aaaaaaaaaa aagctttccc gcggccagct tggcgtaatc
     2281 atggtcatag ctgtttcctg tgtgaaattg ttatccgctg ccaccgctga gcaataacta
     2341 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa agccaattct
     2401 gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     2461 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     2521 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     2581 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     2641 actgaatccg gtgagaatgg caaaagctta tgcatttctt tccagacttg ttcaacaggc
     2701 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     2761 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     2821 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     2881 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     2941 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     3001 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     3061 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     3121 acattatcg
//
