LOCUS       sequence-336156-        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"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     581..600
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        581..599
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        603..652
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             703..725
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             complement(832..1155)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     1065..1081
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1082..1138
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1151..1167)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1175..1191
                     /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(1199..1205,1206..1223,1224..1229))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1244..1265
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1343..1361)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1357..1404
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1574..1590)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1598..1614
                     /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(1622..1628,1629..1646,1647..1652))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1667..1688
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1805..1822)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1976..2564)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2056..2075)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2735..3550)
                     /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     3458..3477
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3551..3653)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     3721..3739
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accaaatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcatcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgcaac gcgatgacga tggatagcga
      421 ttcatcgatg agctgacccg atcgccgccg ccggagggtt gcgtttgaga cgggcgacag
      481 atcttaagca ttactcgcat ccattctcag gctgtctcgt ctcgtctccg gaagacatgc
      541 ttaggagcct gcattaggat cgatctcgat cccgcgaaat taatacgact cactataggg
      601 agctgtcacc ggatgtgctt tccggtctga tgagtccgtg aggacgaaac agcctctaca
      661 aataattttg tttaatacta gacagaaaca gaggagatat gctttgttta actttaagaa
      721 ggagatatac aaatgtgaga ccgtcacagc ttgtctgtaa gcggatgccg ggagcagaca
      781 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
      841 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
      901 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg
      961 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     1021 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     1081 tgaattcgag ctcggtaccc ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg
     1141 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     1201 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     1261 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctag
     1321 gtctcagctt aggtcgccta ggccgctgag caataactag cataacccct tggggcctct
     1381 aaacgggtct tgaggggttt tttgctgaaa acctcgcttt gtcatgtctt caggctggga
     1441 gttcgtagac ggaaacaaac gcagaatcca agccgcgcgc ggccaggata catagattac
     1501 cacaactccg agcccttcca cccgcatcag ttctggacca gcgagctgtg ctgcgactcg
     1561 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     1621 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     1681 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     1741 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     1801 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     1861 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     1921 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     1981 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2041 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2101 ctgttccgac cctgtcgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2161 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2221 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     2281 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     2341 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     2401 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     2461 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     2521 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     2581 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     2641 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     2701 tctaaagtat atatgagtaa acttggtctg acagttagaa aaactcatcg agcatcaagt
     2761 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     2821 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     2881 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     2941 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaagagtt
     3001 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     3061 tcgcaccaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     3121 cgccgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     3181 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     3241 tccctgggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     3301 tggtcggaag aggcataaat tccgtcagcc agtttagcct gaccatctca tctgtaacat
     3361 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     3421 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     3481 ataaatcagc atccatgttg gaatttaatc gcggcttcaa gcaagacgtt tcccgttgaa
     3541 tatggctcat tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg
     3601 gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga tcacacattt
     3661 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     3721 aataggcgta tcacgaggcc ctttcgtc
//
