LOCUS       sequence-320849-        2370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Acceptor for golden gate assembly with BsaI overhangs GCTT-AATG, 
            adds a N-terminal expression tag. Generates plasmids for T7-driven 
            E. coli cell-free protein synthesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2370)
  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 2370)
  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: pEPQD0KN0024
FEATURES             Location/Qualifiers
     source          1..2370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     36..55
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        36..54
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             86..108
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             complement(230..553)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     235..254
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     448..470
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     462..479
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     463..479
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    480..536
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(549..565)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(549..565)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(562..584)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    573..589
                     /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(597..603,604..621,622..627))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    642..663
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(774..792)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      788..835
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(853..1668)
                     /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     1576..1595
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1660..1679
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(1790..2370,1..8)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2279..2298
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatggaaaaa cgccagcaac gcgatcccgc gaaattaata cgactcacta tagggacacc
       61 acaacggttt ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgga
      121 gaaaaaaata atgtgagacc gtcacagctt gtctgtaagc ggatgccggg agcagacaag
      181 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
      241 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
      301 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
      361 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
      421 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
      481 aattcgagct cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggcg
      541 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
      601 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
      661 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctaggt
      721 ctcagcttcg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
      781 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
      841 agccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
      901 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
      961 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     1021 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     1081 atgagtgacg actgaatccg gtgagaatgg caaaagctta tgcatttctt tccagacttg
     1141 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     1201 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     1261 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     1321 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     1381 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     1441 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     1501 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     1561 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     1621 atttaatcgc ggcttcgagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     1681 attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc
     1741 aatgtaacat cagagatttt gagacacaac gtgagatcaa aggatcttct tgagatcctt
     1801 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1861 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1921 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     1981 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2041 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2101 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2161 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2221 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2281 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2341 ttttgtgatg ctcgtcaggg gggcggagcc
//
