LOCUS       sequence-452394-        3067 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTet-driven expression of F30-scaffolded Pepper aptamer and 
            mTurquoise2 for quantifying cell-free RNA and protein synthesis. The
            mTurq2 has a GGGGS linker followed by a His6x tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3067)
  AUTHORS   Kapasiawala M, Murray RM
  TITLE     Metabolic perturbations to an E. coli-based cell-free system reveal 
            a trade-off between transcription and translation
  JOURNAL   bioRxiv 2023.03.22.533877
REFERENCE   2  (bases 1 to 3067)
  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: "bioRxiv 
            2023.03.22.533877"
COMMENT     Sequence Label: PTet-F30-Pepper aptamer-UTR1-mTurq2
FEATURES             Location/Qualifiers
     source          1..3067
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    5..23
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    30..48
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             170..192
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             932..949
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    1059..1079
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1080..1137
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(1161..1178)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(1386..1405)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1397..2212
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1470..1489)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2334..2922)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2414..2433)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2963..2981
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2985..3028)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    3031..3052
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
ORIGIN
        1 ggagtcccta tcagtgatag agattgacat ccctatcagt gatagagata ctgagcacta
       61 ctttgccatg tgtatgtggg ttcgcccaca tactctgatg atccccaatc gtggcgtgtc
      121 ggcctgcttc ggcaggcact ggcgccggga tcattcatgg caaaataatt ttgtttaact
      181 ttaagaagga gatataccaa tggtaagtaa gggcgaagag ttgtttactg gggtagtccc
      241 gatcctggtc gagctggatg gggacgttaa cggccataaa tttagcgtgt cgggtgaagg
      301 cgagggggac gcgacctacg ggaaactgac cttgaagttt atctgtacga ccgggaaact
      361 tccggtgcca tggcctacac tcgtcacgac cttgagctgg ggtgttcaat gctttgcacg
      421 ttacccagac catatgaaac agcatgattt cttcaaaagt gcaatgccag aagggtatgt
      481 tcaagagcgc accatctttt ttaaggacga tggcaactat aagacccgtg ccgaagtcaa
      541 gttcgaaggt gacacccttg tcaaccgtat cgagttgaaa ggtatcgact ttaaggaaga
      601 tgggaatatc ctgggtcata aacttgaata taattacttt tccgacaatg tttatatcac
      661 agccgacaaa caaaaaaacg gtatcaaggc caatttcaag attcggcata acatcgaaga
      721 tgggggggtt caactcgctg atcattacca gcagaataca cctatcggcg acggcccagt
      781 cttattacct gataatcatt accttagcac ccaatccaaa ctcagcaagg atccaaacga
      841 aaagcgcgat cacatggtgc ttttggagtt tgtcactgct gctggtatta cattgggtat
      901 ggatgaattg tacaaagggg gcggcggtag ccatcatcat catcaccatt aaaggtttca
      961 gccaaaaaac ttaagaccgc cggtcttgtc cactaccttg cagtaatgcg gtggacagga
     1021 tcggcggttt tcttttctct tctcaagctt atgtcttcta ctagtagcgg ccgctgcagt
     1081 ccggcaaaaa agggcaaggt gtcaccaccc tgcccttttt ctttaaaacc gaaaagatta
     1141 cttcgcgtta tgcaggcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     1201 cgagcggtat cagctcactc aaaggcggta agtcgcgtgt tatacgcccg ttatccatgg
     1261 gtatggacag ttttcccttt gatatgtaac gcacgttgtg tctcaaaatc tctgatgtta
     1321 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     1381 taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctccc gtccgcgctt
     1441 aaactccaac atggacgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     1501 atcaggtgcg acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa
     1561 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtccgtc tcaactggct
     1621 gacggagttt atgcctctcc cgaccatcaa gcattttatc cgtactcctg atgatgcgtg
     1681 gttactcacc accgcgattc ctgggaaaac agccttccag gtattagaag aatatcctga
     1741 ttcaggtgaa aatattgttg atgcgctggc cgtgttcctg cgccggttac attcgattcc
     1801 tgtttgtaat tgtcctttta acagcgatcg tgtatttcgt cttgctcagg cgcaatcacg
     1861 catgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     1921 tgaacaagtc tggaaagaaa tgcacaagct cttgccattc tcaccggatt cagtcgtcac
     1981 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     2041 tgatgttgga cgggtcggaa tcgcagaccg ttaccaggac cttgccattc tttggaactg
     2101 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     2161 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aataatacta
     2221 gcagaaatca tccttagcga aagctaagga ttttttttat ctgattaccg cctttgagtg
     2281 agcgtcgacc tagtcctagc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2341 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2401 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2461 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2521 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2581 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2641 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2701 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2761 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2821 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2881 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2941 tctacggggt ctgacgcgca taaataggcg tatcacgagg cagaatttca gataaaaaaa
     3001 atccttagct ttcgctaagg atgatttctg gaattcgcgg ccgcttctag agactagtgg
     3061 aagacat
//
