LOCUS       sequence-345640-        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(16..33)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(187..775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(267..286)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(946..1761)
                     /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     1669..1688
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1762..1864)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     1932..1950
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1988..2010)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2110..2129
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2323..2345
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2337..2354
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2338..2354
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2540..2559
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2540..2558
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        2562..2611
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             2662..2684
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2692..3402
                     /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(3419..3437)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3433..3480
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3650..3666)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3650..3666)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3663..3685)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3674..3690
                     /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(3698..3704,3705..3722,3723..3728))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3743..3764
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
       61 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
      121 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
      181 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
      241 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
      301 cgtgcgctct cctgttccga ccctgtcgct taccggatac ctgtccgcct ttctcccttc
      361 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
      421 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
      481 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
      541 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
      601 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
      661 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
      721 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
      781 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
      841 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
      901 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttaga aaaactcatc
      961 gagcatcaag tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     1021 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     1081 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     1141 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     1201 tggcaagagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     1261 atcaaaatca ctcgcaccaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     1321 aaatacgcga tcgccgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     1381 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     1441 gaatgctgtt ttccctggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     1501 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagcc tgaccatctc
     1561 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     1621 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     1681 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcttca agcaagacgt
     1741 ttcccgttga atatggctca ttttagcttc cttagctcct gaaaatctcg ataactcaaa
     1801 aaatacgccc ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccg
     1861 atcacacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     1921 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     1981 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     2041 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     2101 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccaaatgcgg tgtgaaatac
     2161 cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca
     2221 actgttggga agggcgatcg gtgcgggcct catcgctatt acgccagctg gcgaaagggg
     2281 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     2341 aaacgacggc cagtgcaacg cgatgacgat ggatagcgat tcatcgatga gctgacccga
     2401 tcgccgccgc cggagggttg cgtttgagac gggcgacaga tcttaagcat tactcgcatc
     2461 cattctcagg ctgtctcgtc tcgtctccgg aagacatgct taggagcctg cattaggatc
     2521 gatctcgatc ccgcgaaatt aatacgactc actataggga gctgtcaccg gatgtgcttt
     2581 ccggtctgat gagtccgtga ggacgaaaca gcctctacaa ataattttgt ttaatactag
     2641 acagaaacag aggagatatg ctttgtttaa ctttaagaag gagatataca aatggtgagc
     2701 aagggcgagg aaaccacaat gggcgtaatc aagcccgaca tgaagatcaa gctgaagatg
     2761 gagggcaacg tgaatggcca cgccttcgtg atcgagggcg agggcgaggg caagccctac
     2821 gacggcacca acaccatcaa cctggaggtg aaggagggag cccccctgcc cttctcctac
     2881 gacattctga ccaccgcgtt cgcctacggc aacagggcct tcaccaagta ccccgacgac
     2941 atccccaact acttcaagca gtccttcccc gagggctact cttgggagcg caccatgacc
     3001 ttcgaggaca agggcatcgt gaaggtgaag tccgacatct ccatggagga ggactccttc
     3061 atctacgaga tacacctcaa gggcgagaac ttccccccca acggccccgt gatgcagaag
     3121 aaaaccaccg gctgggacgc ctccaccgag aggatgtacg tgcgcgacgg cgtgctgaag
     3181 ggcgacgtca agcacaagct gctgctggag ggcggcggcc accaccgcgt tgacttcaag
     3241 accatctaca gggccaagaa ggcggtgaag ctgcccgact atcactttgt ggaccaccgc
     3301 atcgagatcc tgaaccacga caaggactac aacaaggtga ccgtttacga gagcgccgtg
     3361 gcccgcaact ccaccgacgg catggacgag ctgtacaagt aagcttaggt cgcctaggcc
     3421 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     3481 ctgaaaacct cgctttgtca tgtcttcagg ctgggagttc gtagacggaa acaaacgcag
     3541 aatccaagcc gcgcgcggcc aggatacata gattaccaca actccgagcc cttccacccg
     3601 catcagttct ggaccagcga gctgtgctgc gactcgtggc gtaatcatgg tcatagctgt
     3661 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     3721 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     3781 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     3841 cggggagagg cggtttgcgt attgg
//
