LOCUS       sequence-177874-        3736 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of zmADH mutant V86TAG for ncAA incoporation using the 
            p70a promoter and UTR1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3736)
  AUTHORS   Schlesinger O, Chemla Y, Heltberg M, Ozer E, Marshall R, Noireaux V,
            Jensen MH, Alfonta L
  TITLE     Tuning of Recombinant Protein Expression in Escherichia coli by 
            Manipulating Transcription, Translation Initiation Rates, and 
            Incorporation of Noncanonical Amino Acids.
  JOURNAL   ACS Synth Biol. 2017 Mar 9. doi: 10.1021/acssynbio.7b00019.
  PUBMED    28230975
REFERENCE   2  (bases 1 to 3736)
  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: "ACS Synth 
            Biol. 2017 Mar 9. doi: 10.1021/acssynbio.7b00019."
COMMENT     Sequence Label: pBEST-p70a-UTR1-NTerm 6xHis-zmADH V86TAG-T500
FEATURES             Location/Qualifiers
     source          1..3736
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             303..325
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             344..361
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             371..388
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1720..1737)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1891..2479)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1971..1990)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2650..3441,3442..3510))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3273..3292
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3511..3615)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3683..3701
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ccagccagaa aacgaccttt ctgtggtgaa accggatgct gcaattcaga gcggcagcaa
       61 gtgggggaca gcagaagacc tgaccgccgc agagtggatg tttgacatgg tgaagactat
      121 cgcaccatca gccagaaaac cgaattttgc tgggtgggct aacgatatcc gcctgatgcg
      181 tgaacgtgac ggacgtaacc accgcgacat gtgtgtgctg ttccgctggg catgctgagc
      241 taacaccgtg cgtgttgaca attttacctc tggcggtgat aatggttgca gctagcaata
      301 attttgttta actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca
      361 cagcagcggc ctggtgccgc gcggcagcca tatggcttct tcaacttttt atattccttt
      421 cgtcaacgaa atgggcgaag gttcgcttga aaaagcaatc aaggatctta acggcagcgg
      481 ctttaaaaat gccctgatcg tttctgatgc tttcatgaac aaatccggtg ttgtgaagca
      541 ggttgctgac ctgttgaaaa cacagggtat taattctgct gtttatgatg gcgttatgcc
      601 gaacccgact gttaccgcag ttctggaagg ccttaagatc ctgaaggata acaattcaga
      661 cttcgtcatc tccctcggtg gtggttctcc ccatgactgc gccaaagcca tcgctctggt
      721 cgcaaccaat ggtggtgaag tcaaagacta cgaaggtatc gacaaatcta agaaacctgc
      781 cctgcctttg atgtcaatca acacgacggc tggtacggct tctgaaatga cgcgtttctg
      841 catcatcact gatgaagtcc gtcacgttaa gatggccatt gttgaccgtc acgttacccc
      901 gatggtttcc gtcaacgatc ctctgttgat ggttggtatg ccaaaaggcc tgaccgccgc
      961 caccggtatg gatgctctga cccacgcatt tgaagcttat tcttcaacgg cagctactcc
     1021 gatcaccgat gcttgcgctt tgaaagcagc ttccatgatc gctaagaatc tgaagaccgc
     1081 ttgcgacaac ggtaaggata tgccagctcg tgaagctatg gcttatgccc aattcctcgc
     1141 tggtatggcc ttcaacaacg cttcgcttgg ttatgtccat gctatggctc accagttggg
     1201 cggttactac aacctgccgc atggtgtctg caacgctgtt ctgcttccgc atgttctggc
     1261 ttataacgcc tctgtcgttg ctggtcgtct gaaagacgtt ggtgttgcta tgggtctcga
     1321 tatcgccaat ctcggcgata aagaaggcgc agaagccacc attcaggctg ttcgcgatct
     1381 ggctgcttcc attggtattc cagcaaatct gaccgagctg ggtgctaaga aagaagatgt
     1441 gccgcttctt gctgaccacg ctctgaaaga tgcttgtgct ctgaccaacc cgcgtcaggg
     1501 tgatcagaaa gaagttgaag aactcttcct gagcgctttc taactcgagc aaagcccgcc
     1561 gaaaggcggg cttttctgtg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc
     1621 ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg
     1681 caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg
     1741 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     1801 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     1861 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     1921 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     1981 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2041 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag
     2101 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     2161 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     2221 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     2281 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     2341 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     2401 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     2461 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     2521 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     2581 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     2641 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     2701 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     2761 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     2821 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     2881 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     2941 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3001 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3061 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     3121 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     3181 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     3241 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     3301 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     3361 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     3421 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     3481 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     3541 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     3601 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     3661 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt
     3721 ctggcgaatc ctctga
//
