LOCUS       sequence-320859-        3126 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 3126)
  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 3126)
  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: pEPQD0KN0284
FEATURES             Location/Qualifiers
     source          1..3126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..35
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     27..44
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     28..44
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    45..101
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(114..130)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    138..154
                     /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(162..168,169..186,187..192))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    207..228
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(947..963)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(987..1005)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1001..1048
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1066..1881)
                     /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     1789..1808
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1873..1892
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2003..2591
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2492..2511
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2710..2728
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2710..2727
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(join(2921..3126,1..118))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     2926..2945
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta
       61 cccggggatc ctctagagtc gacctgcagg catgcaagct tggcgtaatc atggtcatag
      121 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
      181 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
      241 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc taggtctcag cttcgggtta
      301 taattacctc aggtcgacgt cccatggttt tgtatagaat ttacggctag cgccggatgc
      361 gacgccggtc gcgtcttatc cggccttcct atatcaggcg gtgtttaaga cgccgccgct
      421 tcgcccaaat ccttatgccg gttcgacgac tggacaaaat actgtttatc ttcccagcgc
      481 aggcaggtta atgtaccacc ccagcagcag ccggtatcca gcgcgtatat accttccggc
      541 gtacctttgc cctccagcga tgcccagtga ccaaaggcga tgctgtattc ttcagcgaca
      601 gggccaggaa tcgcaaacca cggtttcagt ggggcagggg cctcttccgg cgattcttac
      661 tagctagtat gcataggtgc tgaaatataa agtttgtgtt tctaaaacac acgtggtacg
      721 tacgataacg tacagtgttt ttccctccac ttaaatcgaa gggtagtgtc ttggagcgcg
      781 cggagtaaac atatatggtt catatatgtc cgtaggcacg taaaaaaagc gagggattcg
      841 aattcccccg gaacccccgg ttggggccca cgcctcgatc gagcaaaaaa aaaaaaagaa
      901 aaaaaaaaaa aaaaagcttt cccgcggcca gcttggcgta atcatggtca tagctgtttc
      961 ctgtgtgaaa ttgttatccg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1021 ctctaaacgg gtcttgaggg gttttttgct gaaagccaat tctgattaga aaaactcatc
     1081 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     1141 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     1201 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     1261 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     1321 tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     1381 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     1441 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     1501 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     1561 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     1621 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     1681 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     1741 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     1801 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcttcg agcaagacgt
     1861 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     1921 tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac
     1981 aacgtgagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2041 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2101 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     2161 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2221 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2281 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2341 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2401 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2461 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2521 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2581 gcctatggaa aaacgccagc aacgcgatcc cgcgacgaat aagccagatg ctacacaatt
     2641 aggcttgtac atactgtcgt tagaacgcgg ctacaattaa tacataacct tatgtatcat
     2701 acacatacga tttaggtgac actatagaac tcacctatct ccccaacacc taataacatt
     2761 caatcactct ttccactaac cacctatcta catcaccaag atatcactag ttctcgagct
     2821 aatgagagac cgtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     2881 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     2941 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     3001 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     3061 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     3121 gggtaa
//
