LOCUS       sequence-338672-        3563 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive T7 RNAP-mediated expression of 6xHis-mCherry with 
            Broccoli RNA aptamer..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3563)
  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 3563)
  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 Broccoli
FEATURES             Location/Qualifiers
     source          1..3563
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..34
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        15..33
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             65..87
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             106..123
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             136..156
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             157..864
                     /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(302..320)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     570..589
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1033..1051)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1047..1094
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1139..1157)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1225..1329
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1330..1398,1399..2190)
                     /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(1548..1567)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2361..2949
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2850..2869
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3103..3120
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3200..3219)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgatcccgcg aaattaatac gactcactat agggagacca caacggtttc cctctagaaa
       61 taattttgtt taactttaag aaggagatat accatgtctg gttctcatca tcatcatcat
      121 catggtagca gcggcgaaaa cctgtatttt cagtccatgg tgagcaaggg cgaggaggat
      181 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
      241 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
      301 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
      361 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
      421 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
      481 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
      541 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
      601 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
      661 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
      721 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc
      781 cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc
      841 ggcggcatgg acgagctgta caagggcagc taaagggtga tccggttgcc atgtgtatgt
      901 gggagacggt cgggtccaga tattcgtatc tgtcgagtag agtgtgggct cccacatact
      961 ctgatgatcc ttcgggatca ttcatggcaa ctgctaacaa agcccgaaag gaagctgagt
     1021 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     1081 tgaggggttt tttgctgaaa ggaggaacta tatccggata attcttgaag acgaaagggc
     1141 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     1201 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1261 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1321 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1381 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1441 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1501 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1561 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     1621 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     1681 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     1741 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     1801 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     1861 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     1921 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     1981 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2041 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2101 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2161 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2221 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2281 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2341 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2401 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2461 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     2521 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2581 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2641 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2701 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2761 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2821 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2881 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     2941 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3001 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3061 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3121 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3181 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     3241 tccgctatcg ctacgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     3301 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     3361 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     3421 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     3481 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca
     3541 ggacccaacg ctgcccgaga tct
//
