LOCUS       sequence-320855-        2354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Acceptor for golden gate assembly with BsaI overhangs GCTT-CCAT. 
            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 2354)
  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 2354)
  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: pEPQD0KN0245
FEATURES             Location/Qualifiers
     source          1..2354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(92..415)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     97..116
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     310..332
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     324..341
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     325..341
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    342..398
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(411..427)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(411..427)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(424..446)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    435..451
                     /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(459..465,466..483,484..489))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    504..525
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(636..654)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      650..697
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(715..1530)
                     /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     1438..1457
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1522..1541
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1652..2240
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2141..2160
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2268..2287
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2268..2286
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2318..2340
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ccgtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
       61 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
      121 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
      181 ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
      241 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
      301 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattcgag ctcggtaccc
      361 ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg
      421 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
      481 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
      541 ctgcccgctt tccagtcggg aaacctgtcg tgccagctag gtctcagctt cggctgctaa
      601 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
      661 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaagccaatt ctgattagaa
      721 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
      781 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
      841 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
      901 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
      961 cggtgagaat ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt
     1021 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     1081 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     1141 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     1201 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     1261 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     1321 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     1381 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     1441 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcttcga
     1501 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     1561 agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt
     1621 ttgagacaca acgtgagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1681 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1741 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1801 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1861 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1921 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1981 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     2041 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     2101 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2161 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     2221 gggggcggag cctatggaaa aacgccagca acgcgatccc gcgaaattaa tacgactcac
     2281 tatagggaca ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga
     2341 tataaccatt gaga
//
