LOCUS       sequence-321454-        4281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Acceptor for golden gate assembly with BsaI overhangs GCTT-CCAT. 
            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 4281)
  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 4281)
  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: pEPQD0CB0246
FEATURES             Location/Qualifiers
     source          1..4281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        241..259
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     241..258
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(452..775)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     670..692
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     684..701
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     685..701
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    702..758
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(771..787)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    795..811
                     /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(819..825,826..843,844..849))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    864..885
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1608..1624)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1632..1648
                     /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(1656..1662,1663..1680,1681..1686))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1701..1722
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2010..2598)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2090..2109)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2769..3560,3561..3629))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIRYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3392..3411
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3630..3734)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3802..3820
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3858..3880)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4213..4232
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ggcgtagagg atctggctag cgatgaccct gctgattggt tcgctgacca tttccgggtg
       61 cgggacggcg ttaccagaaa ctcagaaggt tcgtccaacc aaaccgactc tggcggcagt
      121 ttacgagaga gatgataggg tctgcttcag taagccagat gctacacaat taggcttgta
      181 catactgtcg ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg
      241 atttaggtga cactatagaa ctcacctatc tccccaacac ctaataacat tcaatcactc
      301 tttccactaa ccacctatct acatcaccaa gatatcacta gttctcgagc tccattgaga
      361 ccgtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
      421 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
      481 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
      541 ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
      601 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
      661 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattcgag ctcggtaccc
      721 ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg
      781 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
      841 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
      901 ctgcccgctt tccagtcggg aaacctgtcg tgccagctag gtctcagctt cgggttataa
      961 ttacctcagg tcgacgtccc atggttttgt atagaattta cggctagcgc cggatgcgac
     1021 gccggtcgcg tcttatccgg ccttcctata tcaggcggtg tttaagacgc cgccgcttcg
     1081 cccaaatcct tatgccggtt cgacgactgg acaaaatact gtttatcttc ccagcgcagg
     1141 caggttaatg taccacccca gcagcagccg gtatccagcg cgtatatacc ttccggcgta
     1201 cctttgccct ccagcgatgc ccagtgacca aaggcgatgc tgtattcttc agcgacaggg
     1261 ccaggaatcg caaaccacgg tttcagtggg gcaggggcct cttccggcga ttcttactag
     1321 ctagtatgca taggtgctga aatataaagt ttgtgtttct aaaacacacg tggtacgtac
     1381 gataacgtac agtgtttttc cctccactta aatcgaaggg tagtgtcttg gagcgcgcgg
     1441 agtaaacata tatggttcat atatgtccgt aggcacgtaa aaaaagcgag ggattcgaat
     1501 tcccccggaa cccccggttg gggcccacgc ctcgatcgag caaaaaaaaa aaaaaagaaa
     1561 aaaaaaaaaa aaaaaagctt tcccgcggcc agcttggcgt aatcatggtc atagctgttt
     1621 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     1681 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     1741 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     1801 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cactcactga ctcgctgcgc
     1861 tcggtcgctc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     1921 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     1981 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2041 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2101 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2161 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2221 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2281 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2341 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2401 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     2461 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     2521 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2581 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     2641 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     2701 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2761 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2821 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2881 tctggcccca gtgctgcaat gataccgcga gatccacgct caccggctcc agatttatca
     2941 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3001 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3061 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3121 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3181 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3241 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3301 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagcg tatgcggcga
     3361 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3421 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3481 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3541 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3601 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     3661 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3721 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     3781 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     3841 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     3901 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     3961 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattcgacg
     4021 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     4081 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     4141 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     4201 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     4261 gatgccggcc acgatgcgtc c
//
