LOCUS       sequence-197052-        3232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of deGFP mutant Y35TAG 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 3232)
  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 3232)
  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-deGFP Y35TAG-6xHis-T500
FEATURES             Location/Qualifiers
     source          1..3232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             231..253
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(290..311)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(551..570)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     898..919
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             944..961
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1144..1161)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1315..1903)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1395..1414)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2074..2865,2866..2934))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2697..2716
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2935..3039)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3107..3125
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 agaagacctg accgccgcag agtggatgtt tgacatggtg aagactatcg caccatcagc
       61 cagaaaaccg aattttgctg ggtgggctaa cgatatccgc ctgatgcgtg aacgtgacgg
      121 acgtaaccac cgcgacatgt gtgtgctgtt ccgctgggca tgctgagcta acaccgtgcg
      181 tgttgacaat tttacctctg gcggtgataa tggttgcagc tagcaataat tttgtttaac
      241 tttaagaagg agatatacca tggagctttt cactggcgtt gttcccatcc tggtcgagct
      301 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      361 ctagggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      421 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
      481 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
      541 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
      601 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
      661 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
      721 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
      781 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
      841 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
      901 ggtcctgctg gagttcgtga ccgccgccgg gatctctaga gtgcaccacc accaccatca
      961 cgtgtaactc gagcaaagcc cgccgaaagg cgggcttttc tgtgtcgacc gatgcccttg
     1021 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca
     1081 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctcttccgc
     1141 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     1201 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     1261 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     1321 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     1381 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1441 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     1501 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1561 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1621 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     1681 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     1741 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     1801 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     1861 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     1921 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     1981 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2041 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     2101 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     2161 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     2221 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     2281 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     2341 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     2401 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     2461 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     2521 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     2581 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     2641 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa
     2701 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     2761 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     2821 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     2881 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     2941 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     3001 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     3061 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     3121 gaggcccttt cgtcttcaag aattctggcg aatcctctga ccagccagaa aacgaccttt
     3181 ctgtggtgaa accggatgct gcaattcaga gcgccagcaa gtgggggaca gc
//
