LOCUS       sequence-320851-        2372 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Acceptor for golden gate assembly with BsaI overhangs GCTT-CCAT, 
            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 2372)
  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 2372)
  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: pEPQD0KN0244
FEATURES             Location/Qualifiers
     source          1..2372
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..49
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        30..48
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             80..102
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             complement(226..549)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     231..250
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     444..466
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     458..475
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     459..475
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    476..532
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(545..561)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(545..561)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(558..580)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    569..585
                     /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(593..599,600..617,618..623))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    638..659
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(770..788)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      784..831
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(849..1664)
                     /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     1572..1591
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1656..1675
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(1786..2372,1..2)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2275..2294
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaaacgccag caacgcgatc ccgcgaaatt aatacgactc actataggga caccacaacg
       61 gtttccctct agaaataatt ttgtttaact ttaagaagga gatatacata tggagaaaaa
      121 aatatccatt gagaccgtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
      181 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
      241 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
      301 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
      361 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
      421 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
      481 cgagctcggt acccggggat cctctagagt cgacctgcag gcatgcaagc ttggcgtaat
      541 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
      601 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
      661 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctaggtctca
      721 gcttcggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
      781 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagcc
      841 aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
      901 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
      961 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     1021 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     1081 gtgacgactg aatccggtga gaatggcaaa agcttatgca tttctttcca gacttgttca
     1141 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     1201 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     1261 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     1321 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     1381 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     1441 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     1501 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     1561 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     1621 aatcgcggct tcgagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     1681 ctgtttatgt aagcagacag ttttattgtt catgatgata tatttttatc ttgtgcaatg
     1741 taacatcaga gattttgaga cacaacgtga gatcaaagga tcttcttgag atcctttttt
     1801 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     1861 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     1921 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     1981 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2041 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2101 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2161 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2221 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2281 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2341 gtgatgctcg tcaggggggc ggagcctatg ga
//
