LOCUS       sequence-338669-        3692 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive T7 RNAP-mediated expression of 6xHis-mCherry with 2xd 
            Broccoli RNA aptamer..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3692)
  AUTHORS   Gonzales DT, Yandrapalli N, Robinson T, Zechner C, Tang TD
  TITLE     Cell-Free Gene Expression Dynamics in Synthetic Cell Populations.
  JOURNAL   ACS Synth Biol. 2022 Jan 21;11(1):205-215. doi: 
            10.1021/acssynbio.1c00376. Epub 2022 Jan 4.
  PUBMED    35057626
REFERENCE   2  (bases 1 to 3692)
  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"; doi: 
            "10.1021/acssynbio.1c00376"; journalName: "ACS Synth Biol"; date: 
            "2022-01-21- 21"; volume: "11"; issue: "1"; pages: "205-215"
COMMENT     Sequence Label: pEXP5-NT/6xHis mCherry F30 2xdBroccoli
FEATURES             Location/Qualifiers
     source          1..3692
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(83..101)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        169..273
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(274..342,343..1134)
                     /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     complement(492..511)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1305..1893
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1794..1813
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2047..2064
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2144..2163)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2522..2541
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2522..2540
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2572..2594
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2613..2630
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2643..2663
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2664..3371
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2809..2827)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3077..3096
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3669..3687)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(3683..3692,1..38)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
       61 gataattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
      121 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
      181 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
      241 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
      301 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
      361 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
      421 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
      481 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
      541 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
      601 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
      661 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
      721 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
      781 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
      841 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
      901 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
      961 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     1021 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     1081 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     1141 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     1201 atctaggtga agatcctttt tgataatctt atgaccaaaa tcccttaacg tgagttttcg
     1261 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     1321 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     1381 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     1441 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     1501 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     1561 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     1621 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     1681 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     1741 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     1801 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     1861 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     1921 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     1981 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     2041 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     2101 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     2161 ccgcatagtt aagccagtat acactccgct atcgctacgc ttcgttaata cagatgtagg
     2221 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     2281 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     2341 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     2401 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     2461 cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatctcga tcccgcgaaa
     2521 ttaatacgac tcactatagg gagaccacaa cggtttccct ctagaaataa ttttgtttaa
     2581 ctttaagaag gagatatacc atgtctggtt ctcatcatca tcatcatcat ggtagcagcg
     2641 gcgaaaacct gtattttcag tccatggtga gcaagggcga ggaggataac atggccatca
     2701 tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg
     2761 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
     2821 tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg
     2881 gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc
     2941 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga
     3001 cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca
     3061 acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg
     3121 agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga
     3181 aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc
     3241 agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact
     3301 acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg
     3361 agctgtacaa gggcagctaa agggtgatct tgccatgtgt atgtgggaga cggtcgggtc
     3421 catctgagac ggtcgggtcc agatattcgt atctgtcgag tagagtgtgg gctcagatgt
     3481 cgagtagagt gtgggctccc acatactctg atgatccaga cggtcgggtc catctgagac
     3541 ggtcgggtcc agatattcgt atctgtcgag tagagtgtgg gctcagatgt cgagtagagt
     3601 gtgggctgga tcattcatgg caacggctgc taacaaagcc cgaaaggaag ctgagttggc
     3661 tgctgccacc gctgagcaat aactagcata ac
//
