LOCUS       sequence-266366-        2624 bp    DNA     circular SYN 15-APR-2025
DEFINITION  sfGFP driven by a T7 promoter with the tetO sequence 4 bp downstream
            from the promoter start site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2624)
  AUTHORS   McManus JB, Lux MW, Murray RM, Emanuel PA
  TITLE     A method for cost-effective and rapid characterization of engineered
            T7-Based transcription factors by cell-free protein synthesis 
            reveals insights into the regulation of T7 Rna polymerase-driven 
            expression.
  JOURNAL   Arch Biochem Biophys. 2019 Jul 18. pii: S0003-9861(19)30288-7. doi: 
            10.1016/j.abb.2019.07.010.
  PUBMED    31326518
REFERENCE   2  (bases 1 to 2624)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Arch 
            Biochem Biophys. 2019 Jul 18. pii: S0003-9861(19)30288-7. doi: 
            10.1016/j.abb.2019.07.010."
COMMENT     Sequence Label: pY71-tet.I.1
FEATURES             Location/Qualifiers
     source          1..2624
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     6..25
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6..24
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    26..44
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        77..126
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             157..168
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             178..198
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             838..885
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             886..909
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(1077..1892)
                     /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     1800..1819
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1884..1903
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2014..2602
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2503..2522
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctactaata cgactcacta tagggtccct atcagtgata gagacttacc tcttcagagg
       61 tcactgacct gacaagctgt caccggatgt gctttccggt ctgatgagtc cgtgaggacg
      121 aaacagcctc tacaaataat tttgtttaat ctagagaaag aggagaaata ctagatggag
      181 aatttatact tccagagtag caaaggtgaa gaactgttta ccggcgttgt gccgattctg
      241 gtggaactgg atggcgatgt gaacggtcac aaattcagcg tgcgtggtga aggtgaaggc
      301 gatgccacga ttggcaaact gacgctgaaa tttatctgca ccaccggcaa actgccggtg
      361 ccgtggccga cgctggtgac caccctgacc tatggcgttc agtgttttag tcgctatccg
      421 gatcacatga aacgtcacga tttctttaaa tctgcaatgc cggaaggcta tgtgcaggaa
      481 cgtacgatta gctttaaaga tgatggcaaa tataaaacgc gcgccgttgt gaaatttgaa
      541 ggcgataccc tggtgaaccg cattgaactg aaaggcacgg attttaaaga agatggcaat
      601 atcctgggcc ataaactgga atacaacttt aatagccata atgtttatat tacggcggat
      661 aaacagaaaa atggcatcaa agcgaatttt accgttcgcc ataacgttga agatggcagt
      721 gtgcagctgg cagatcatta tcagcagaat accccgattg gtgatggtcc ggtgctgctg
      781 ccggataatc attatctgag cacgcagacc gttctgtcta aagatccgaa cgaaaaacgg
      841 gaccacatgg ttctgcacga atatgtgaat gcggcaggta ttacgtggag ccatccgcag
      901 ttcgaaaaat aataaacaat aactgaatag ggatcccgac tggcgagagc caggtaacga
      961 atggatcctt cagccaaaaa acttaagacc gccggtcttg tccactacct tgcagtaatg
     1021 cggtggacag gatcggcggt tttcttttct cttctcaagc tgaaagccaa ttctgattag
     1081 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     1141 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     1201 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     1261 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     1321 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca
     1381 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     1441 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     1501 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     1561 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     1621 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     1681 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     1741 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     1801 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcttc
     1861 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     1921 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga
     1981 ttttgagaca caacgtgaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2041 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2101 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2161 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2221 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2281 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2341 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2401 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     2461 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2521 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     2581 aggggggcgg agcctatgga aaaacgccag atctcgatcc cgcg
//
