LOCUS       sequence-320854-        2355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Acceptor for golden gate assembly with BsaI overhangs GCTT-AATG. 
            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 2355)
  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 2355)
  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: pEPQD0KN0025
FEATURES             Location/Qualifiers
     source          1..2355
                     /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(215..538)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     220..239
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     433..455
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     447..464
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     448..464
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    465..521
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(534..550)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(534..550)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(547..569)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    558..574
                     /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(582..588,589..606,607..612))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    627..648
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(759..777)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      773..820
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(838..1653)
                     /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     1561..1580
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1645..1664
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(1775..2355,1..8)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2264..2283
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatggaaaaa cgccagcaac gcgatcccgc gaaattaata cgactcacta tagggacacc
       61 acaacggttt ccctctagaa ataattttgt ttaactttaa gaaggagata tacaaatgtg
      121 agaccgtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
      181 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
      241 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
      301 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
      361 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
      421 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta
      481 cccggggatc ctctagagtc gacctgcagg catgcaagct tggcgtaatc atggtcatag
      541 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
      601 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
      661 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc taggtctcag cttcggctgc
      721 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
      781 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaagcca attctgatta
      841 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
      901 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
      961 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     1021 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     1081 atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa caggccagcc
     1141 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     1201 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     1261 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     1321 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     1381 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     1441 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     1501 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     1561 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggctt
     1621 cgagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     1681 agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag
     1741 attttgagac acaacgtgag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     1801 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     1861 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     1921 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     1981 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     2041 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     2101 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     2161 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     2221 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2281 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2341 caggggggcg gagcc
//
