LOCUS       sequence-452402-        3237 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTet-driven expression of MGapt and deGFP for quantifying cell-free 
            RNA and protein synthesis..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3237)
  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 3237)
  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: pBEST-PTet-MGapt-UTR1-deGFP
FEATURES             Location/Qualifiers
     source          1..3237
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     6..24
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     protein_bind    292..310
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    317..335
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             397..419
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(456..477)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(717..736)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1064..1085
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1280..1297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1451..2039)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1531..1550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2210..3001,3002..3070))
                     /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     2833..2852
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3071..3175)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attctggcga atcctctgac
       61 cagccagaaa acgacctttc tgtggtgaaa ccggatgctg caattcagag cgccagcaag
      121 tgggggacag cagaagacct gaccgccgca gagtggatgt ttgacatggt gaagactatc
      181 gcaccatcag ccagaaaacc gaattttgct gggtgggcta acgatatccg cggatcccga
      241 ctgatgcgtg aacgtgacgg acgtaaccac cgcgacatgt gtgtgctgtt ctccctatca
      301 gtgatagaga ttgacatccc tatcagtgat agagatactg agcacgctag cgggatcccg
      361 actggcgaga gccaggtaac gaatggatcc aataattttg tttaacttta agaaggagat
      421 ataccatgga gcttttcact ggcgttgttc ccatcctggt cgagctggac ggcgacgtaa
      481 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
      541 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
      601 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
      661 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
      721 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
      781 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
      841 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
      901 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
      961 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     1021 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1081 tcgtgaccgc cgccgggatc taactcgagc aaagcccgcc gaaaggcggg cttttctgtg
     1141 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     1201 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     1261 ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     1321 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     1381 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     1441 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     1501 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     1561 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     1621 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     1681 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     1741 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     1801 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     1861 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     1921 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     1981 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     2041 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     2101 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     2161 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     2221 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     2281 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     2341 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     2401 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     2461 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     2521 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     2581 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     2641 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     2701 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     2761 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     2821 cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     2881 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     2941 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     3001 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     3061 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     3121 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     3181 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacct
//
