LOCUS       sequence-320856-        2478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Acceptor for golden gate assembly with BsaI overhangs GCTT-AATG. 
            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: pEPQD0KN0282
FEATURES             Location/Qualifiers
     source          1..2478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(76..399)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     81..100
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     294..316
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     308..325
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     309..325
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    326..382
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(395..411)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(395..411)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(408..430)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    419..435
                     /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(443..449,450..467,468..473))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    488..509
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(620..638)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      634..681
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(699..1514)
                     /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     1422..1441
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1506..1525
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1636..2224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2125..2144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2343..2361
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2343..2360
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
       61 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
      121 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt
      181 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
      241 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
      301 acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat cctctagagt
      361 cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
      421 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
      481 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
      541 cgggaaacct gtcgtgccag ctaggtctca gcttcggctg ctaacaaagc ccgaaaggaa
      601 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
      661 cgggtcttga ggggtttttt gctgaaagcc aattctgatt agaaaaactc atcgagcatc
      721 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
      781 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
      841 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
      901 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
      961 agcttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     1021 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     1081 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     1141 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     1201 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     1261 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     1321 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     1381 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     1441 ccatataaat cagcatccat gttggaattt aatcgcggct tcgagcaaga cgtttcccgt
     1501 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     1561 catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga cacaacgtga
     1621 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1681 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1741 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     1801 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1861 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1921 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1981 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2041 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2101 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2161 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     2221 gaaaaacgcc agcaacgcga tcccgcgacg aataagccag atgctacaca attaggcttg
     2281 tacatactgt cgttagaacg cggctacaat taatacataa ccttatgtat catacacata
     2341 cgatttaggt gacactatag aactcaccta tctccccaac acctaataac attcaatcac
     2401 tctttccact aaccacctat ctacatcacc aagatatcac tagttctcga gctaatgaga
     2461 gaccgtcaca gcttgtct
//
