LOCUS       sequence-345639-        3748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pFGC-T7-RJBB to clone genes by Golden Gate (BsaI). To be used in 
            cell-free systems.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3748)
  AUTHORS   Guzman-Chavez F, Arce A, Adhikari A, Vadhin S, Pedroza-Garcia JA, 
            Gandini C, Ajioka JW, Molloy J, Sanchez-Nieto S, Varner JD, Federici
            F, Haseloff J
  TITLE     Constructing Cell-Free Expression Systems for Low-Cost Access.
  JOURNAL   ACS Synth Biol. 2022 Mar 18;11(3):1114-1128. doi: 
            10.1021/acssynbio.1c00342. Epub 2022 Mar 8.
  PUBMED    35259873
REFERENCE   2  (bases 1 to 3748)
  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.1c00342"; journalName: "ACS Synth Biol"; date: 
            "2022-03-18- 18"; volume: "11"; issue: "3"; pages: "1114-1128"
COMMENT     Sequence Label: pT7-RiboJ-LacZa-T7
FEATURES             Location/Qualifiers
     source          1..3748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    20..41
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(158..175)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(329..917)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(409..428)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1088..1903)
                     /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="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1811..1830
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1904..2006)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     2074..2092
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2130..2152)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2480..2496
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2682..2701
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2682..2700
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        2704..2753
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             2804..2826
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             complement(2933..3256)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     3166..3182
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3183..3239
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3252..3268)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3276..3292
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3300..3306,3307..3324,3325..3330))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3345..3366
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3444..3462)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3458..3505
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3675..3691)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3699..3715
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3723..3729,3730..3747,3748,1..5))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
       61 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
      121 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
      181 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
      241 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
      301 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
      361 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
      421 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgtcg cttaccggat
      481 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
      541 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
      601 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
      661 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
      721 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
      781 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
      841 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
      901 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
      961 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     1021 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     1081 ctgacagtta gaaaaactca tcgagcatca agtgaaactg caatttattc atatcaggat
     1141 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     1201 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     1261 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     1321 tgacgactga atccggtgag aatggcaaga gtttatgcat ttctttccag acttgttcaa
     1381 caggccagcc attacgctcg tcatcaaaat cactcgcacc aaccaaaccg ttattcattc
     1441 gtgattgcgc ctgagcgaga cgaaatacgc gatcgccgtt aaaaggacaa ttacaaacag
     1501 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     1561 caggatattc ttctaatacc tggaatgctg ttttccctgg gatcgcagtg gtgagtaacc
     1621 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     1681 gccagtttag cctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     1741 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     1801 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     1861 atcgcggctt caagcaagac gtttcccgtt gaatatggct cattttagct tccttagctc
     1921 ctgaaaatct cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa
     1981 agttggaacc tcttacgtgc cgatcacaca tttccccgaa aagtgccacc tgacgtctaa
     2041 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     2101 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     2161 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     2221 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     2281 caccaaatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     2341 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcatcgcta
     2401 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     2461 ttttcccagt cacgacgttg taaaacgacg gccagtgcaa cgcgatgacg atggatagcg
     2521 attcatcgat gagctgaccc gatcgccgcc gccggagggt tgcgtttgag acgggcgaca
     2581 gatcttaagc attactcgca tccattctca ggctgtctcg tctcgtctcc ggaagacatg
     2641 cttaggagcc tgcattagga tcgatctcga tcccgcgaaa ttaatacgac tcactatagg
     2701 gagctgtcac cggatgtgct ttccggtctg atgagtccgt gaggacgaaa cagcctctac
     2761 aaataatttt gtttaatact agacagaaac agaggagata tgctttgttt aactttaaga
     2821 aggagatata caaatgtgag accgtcacag cttgtctgta agcggatgcc gggagcagac
     2881 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     2941 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
     3001 taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag
     3061 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     3121 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     3181 gtgaattcga gctcggtacc cggggatcct ctagagtcga cctgcaggca tgcaagcttg
     3241 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     3301 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     3361 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagcta
     3421 ggtctcagct taggtcgcct aggccgctga gcaataacta gcataacccc ttggggcctc
     3481 taaacgggtc ttgaggggtt ttttgctgaa aacctcgctt tgtcatgtct tcaggctggg
     3541 agttcgtaga cggaaacaaa cgcagaatcc aagccgcgcg cggccaggat acatagatta
     3601 ccacaactcc gagcccttcc acccgcatca gttctggacc agcgagctgt gctgcgactc
     3661 gtggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3721 cacaacatac gagccggaag cataaagt
//
