LOCUS       sequence-199472-        2483 bp    DNA     circular SYN 14-APR-2025
DEFINITION  In vitro expression of YPet from the T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2483)
  AUTHORS   Stark JC, Huang A, Nguyen PQ, Dubner RS, Hsu KJ, Ferrante TC, 
            Anderson M, Kanapskyte A, Mucha Q, Packett JS, Patel P, Patel R, Qaq
            D, Zondor T, Burke J, Martinez T, Miller-Berry A, Puppala A, 
            Reichert K, Schmid M, Brand L, Hill LR, Chellaswamy JF, Faheem N, 
            Fetherling S, Gong E, Gonzalzles EM, Granito T, Koritsaris J, Nguyen
            B, Ottman S, Palffy C, Patel A, Skweres S, Slaton A, Woods T, 
            Donghia N, Pardee K, Collins JJ, Jewett MC
  TITLE     BioBits Bright: A fluorescent synthetic biology education kit.
  JOURNAL   Sci Adv. 2018 Aug 1;4(8):eaat5107. doi: 10.1126/sciadv.aat5107. 
            eCollection 2018 Aug.
  PUBMED    30083609
REFERENCE   2  (bases 1 to 2483)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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):eaat5107. doi"; pages: " 
            10.1126/sciadv.aat5107. eCollection 2018 Aug"
COMMENT     Sequence Label: pJL1-YPet
FEATURES             Location/Qualifiers
     source          1..2483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             211..233
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(264..283)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(265..283)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      complement(311..899)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(391..410)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1010..1029)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1021..1836
                     /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="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1094..1113)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(1854..1901)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1897..1915
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(join(1967..2483,1..197,198..200,201..203))
                     /codon_start=1
                     /product="yellow fluorescent protein optimized for FRET 
                     with CyPet (Nguyen and Daugherty, 2005)"
                     /label=YPet
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLLCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAAGITEGMNELYK"
     primer_bind     2361..2380
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 ccgtagccca gggtggtcac cagggtgggc cagggcacgg gcagcttgcc ggtggtgcac
       61 agcagcttca gggtcagctt gccgtaggtg gcgtcgccct cgccctcgcc gctcacgctg
      121 aacttgtggc cgttcacgtc gccgtccagc tccaccagga tgggcaccac gccggtgaac
      181 agctcttcgc ctttgctcac catatgtata tctccttctt aaagttaaac aaaattattt
      241 ctagagggaa accgttgtgg tctccctata gtgagtcgta ttaatttcgc gggatcgcgt
      301 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
      361 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
      421 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
      481 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
      541 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
      601 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
      661 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
      721 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
      781 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
      841 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
      901 aagatccttt gatctcacgt tgtgtctcaa aatctctgat gttacattgc acaagataaa
      961 aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     1021 atgagccata ttcaacggga aacgtcttgc tcgaagccgc gattaaattc caacatggat
     1081 gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc
     1141 tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc
     1201 gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct
     1261 cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg
     1321 atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt
     1381 gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct
     1441 tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg
     1501 gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa
     1561 gaaatgcata agcttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca
     1621 cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc
     1681 ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct
     1741 ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa
     1801 ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggcttt cagcaaaaaa
     1861 cccctcaaga cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca
     1921 gcagccaact cagcttcctt tcgggctttg ttagcagccg gtcgacttac ttatagagct
     1981 cgttcatgcc ctcggtgatg ccggcggcgg tcaggaactc cagcagcacc atgtggtccc
     2041 gcttctcgtt ggggtccttg aacagggcgc tctggtagct caggtagtgg ttgtcgggca
     2101 gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtcggcc agctgcacgc
     2161 cgccgtcctc gatgttgtgc cggatcttga agttggcctt gatgccgttc ttctgcttgt
     2221 cggcggtgat gtacacgttg tggctgttgt agttgtactc cagcttgtgg cccaggatgt
     2281 tgccgtcctc cttgaagtcg atgcccttca gctcgatccg gttcaccagg gtgtcgccct
     2341 cgaacttcac ctcggcccgg gtcttgtagt tgccgtcgtc cttgaagaag atggtccgct
     2401 cctgcacgta gccctcgggc atggcgctct tgaagaagtc gtgctgcttc atgtggtcgg
     2461 ggtaccgggc gaagcactgc acg
//
