LOCUS       sequence-224289-        4093 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP (a GFP) in Ecoli off a T7 promoter when exposed to 
            extracted and RPA-amplified kiwi DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4093)
  AUTHORS   Huang A, Nguyen PQ, Stark JC, Takahashi MK, Donghia N, Ferrante T, 
            Dy AJ, Hsu KJ, Dubner RS, Pardee K, Jewett MC, Collins JJ
  TITLE     BioBits Explorer: A modular synthetic biology education kit.
  JOURNAL   Sci Adv. 2018 Aug 1;4(8):eaat5105. doi: 10.1126/sciadv.aat5105. 
            eCollection 2018 Aug.
  PUBMED    30083608
REFERENCE   2  (bases 1 to 4093)
  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: "Sci Adv."; 
            date: "2018-08-1"; volume: "4(8):eaat5105. doi"; pages: " 
            10.1126/sciadv.aat5105. eCollection 2018 Aug"
COMMENT     Sequence Label: pCOLA kiwi sensor sfGFP
FEATURES             Location/Qualifiers
     source          1..4093
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     35..54
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        35..53
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             800..847
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             848..871
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     terminator      875..921
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1151..1966)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1151..1966)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1874..1893
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1967..2058)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2076..2711)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     complement(2221..2240)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2909..3991)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3953..3972
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3992..4069)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gaagtctaac gctgctctgg gctaactgtc gcgctaatac gactcactat agggcgttct
       61 tcatcgatgc gagagccgag atatccgttg ggactttaga acagaggaga taaagatgca
      121 acggatatca aacctggcgg cagcgcaaaa gatgagcaaa ggtgaagaac tgtttaccgg
      181 cgttgtgccg attctggtgg aactggatgg cgatgtgaac ggtcacaaat tcagcgtgcg
      241 tggtgaaggt gaaggcgatg ccacgattgg caaactgacg ctgaaattta tctgcaccac
      301 cggcaaactg ccggtgccgt ggccgacgct ggtgaccacc ctgacctatg gcgttcagtg
      361 ttttagtcgc tatccggatc acatgaaacg tcacgatttc tttaaatctg caatgccgga
      421 aggctatgtg caggaacgta cgattagctt taaagatgat ggcaaatata aaacgcgcgc
      481 cgttgtgaaa tttgaaggcg ataccctggt gaaccgcatt gaactgaaag gcacggattt
      541 taaagaagat ggcaatatcc tgggccataa actggaatac aactttaata gccataatgt
      601 ttatattacg gcggataaac agaaaaatgg catcaaagcg aattttaccg ttcgccataa
      661 cgttgaagat ggcagtgtgc agctggcaga tcattatcag cagaataccc cgattggtga
      721 tggtccggtg ctgctgccgg ataatcatta tctgagcacg cagaccgttc tgtctaaaga
      781 tccgaacgaa aaaggcacgc gggaccacat ggttctgcac gaatatgtga atgcggcagg
      841 tattacgtgg agccatccgc agttcgaaaa ataatagcat aaccccttgg ggcctctaaa
      901 cgggtcttga ggggtttttt gctgaaacct caggcatttg agaagcacac ggtcacactg
      961 cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc
     1021 tgccctgaac cgacgacaag ctgacgaccg ggtctccgca agtggcactt ttcggggaaa
     1081 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1141 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     1201 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     1261 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     1321 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     1381 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     1441 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     1501 ttcgtgattg cgcctgagcg agacgaaata cgcggtcgct gttaaaagga caattacaaa
     1561 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     1621 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     1681 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     1741 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     1801 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     1861 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     1921 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcatactc ttcctttttc
     1981 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     2041 tttagaaaaa taaacaaata ggcatgctag cgcagaaacg tcctagaaga tgccaggagg
     2101 atacttagca gagagacaat aaggccggag cgaagccgtt tttccatagg ctccgccccc
     2161 ctgacgaaca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat
     2221 aaagatacca ggcgtttccc cctgatggct ccctcttgcg ctctcctgtt cccgtcctgc
     2281 ggcgtccgtg ttgtggtgga ggctttaccc aaatcaccac gtcccgttcc gtgtagacag
     2341 ttcgctccaa gctgggctgt gtgcaagaac cccccgttca gcccgactgc tgcgccttat
     2401 ccggtaacta tcatcttgag tccaacccgg aaagacacga caaaacgcca ctggcagcag
     2461 ccattggtaa ctgagaatta gtggatttag atatcgagag tcttgaagtg gtggcctaac
     2521 agaggctaca ctgaaaggac agtatttggt atctgcgctc cactaaagcc agttaccagg
     2581 ttaagcagtt ccccaactga cttaaccttc gatcaaaccg cctccccagg cggttttttc
     2641 gtttacagag caggagatta cgacgatcgt aaaaggatct caagaagatc ctttacggat
     2701 tcccgacacc atcactctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag
     2761 tcagccccat acgatataag ttgtaattct catgttagtc atgccccgcg cccaccggaa
     2821 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     2881 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     2941 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3001 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     3061 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     3121 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     3181 ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     3241 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     3301 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     3361 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     3421 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     3481 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     3541 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     3601 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     3661 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     3721 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     3781 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     3841 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     3901 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     3961 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     4021 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     4081 cgctctccct tat
//
