LOCUS       sequence-336157-        3865 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes mTFP fluorescent protein. To be used in cell-free system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3865)
  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 3865)
  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-mTFP1-T7
FEATURES             Location/Qualifiers
     source          1..3865
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     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             733..1443
                     /codon_start=1
                     /product="monomeric teal (cyan) fluorescent protein (Ai et 
                     al., 2006)"
                     /label=mTFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFAYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKKTTGWDASTERMYVRDGVLK
                     GDVKHKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYESA
                     VARNSTDGMDELYK"
     primer_bind     complement(1460..1478)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1474..1521
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1691..1707)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1691..1707)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1704..1726)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1715..1731
                     /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(1739..1745,1746..1763,1764..1769))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1784..1805
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1922..1939)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2093..2681)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2173..2192)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2852..3667)
                     /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     3575..3594
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3668..3770)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     3838..3856
                     /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 aaatggtgag caagggcgag gaaaccacaa tgggcgtaat caagcccgac
      781 atgaagatca agctgaagat ggagggcaac gtgaatggcc acgccttcgt gatcgagggc
      841 gagggcgagg gcaagcccta cgacggcacc aacaccatca acctggaggt gaaggaggga
      901 gcccccctgc ccttctccta cgacattctg accaccgcgt tcgcctacgg caacagggcc
      961 ttcaccaagt accccgacga catccccaac tacttcaagc agtccttccc cgagggctac
     1021 tcttgggagc gcaccatgac cttcgaggac aagggcatcg tgaaggtgaa gtccgacatc
     1081 tccatggagg aggactcctt catctacgag atacacctca agggcgagaa cttccccccc
     1141 aacggccccg tgatgcagaa gaaaaccacc ggctgggacg cctccaccga gaggatgtac
     1201 gtgcgcgacg gcgtgctgaa gggcgacgtc aagcacaagc tgctgctgga gggcggcggc
     1261 caccaccgcg ttgacttcaa gaccatctac agggccaaga aggcggtgaa gctgcccgac
     1321 tatcactttg tggaccaccg catcgagatc ctgaaccacg acaaggacta caacaaggtg
     1381 accgtttacg agagcgccgt ggcccgcaac tccaccgacg gcatggacga gctgtacaag
     1441 taagcttagg tcgcctaggc cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1501 cgggtcttga ggggtttttt gctgaaaacc tcgctttgtc atgtcttcag gctgggagtt
     1561 cgtagacgga aacaaacgca gaatccaagc cgcgcgcggc caggatacat agattaccac
     1621 aactccgagc ccttccaccc gcatcagttc tggaccagcg agctgtgctg cgactcgtgg
     1681 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     1741 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     1801 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     1861 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     1921 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     1981 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2041 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2101 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2161 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2221 ttccgaccct gtcgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2281 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2341 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2401 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2461 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2521 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2581 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2641 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2701 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2761 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     2821 aaagtatata tgagtaaact tggtctgaca gttagaaaaa ctcatcgagc atcaagtgaa
     2881 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     2941 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     3001 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     3061 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aagagtttat
     3121 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     3181 caccaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     3241 cgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3301 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     3361 ctgggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3421 tcggaagagg cataaattcc gtcagccagt ttagcctgac catctcatct gtaacatcat
     3481 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3541 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3601 aatcagcatc catgttggaa tttaatcgcg gcttcaagca agacgtttcc cgttgaatat
     3661 ggctcatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta
     3721 gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca cacatttccc
     3781 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     3841 aggcgtatca cgaggccctt tcgtc
//
