LOCUS       sequence-389806-        2709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP under the control of PR promoter, p15A origin of 
            replication, Chloramphenicol selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2709)
  AUTHORS   Sarkar K, Mukhopadhyay S, Bonnerjee D, Srivastava R, Bagh S
  TITLE     A frame-shifted gene, which rescued its function by non-natural 
            start codons and its application in constructing synthetic gene 
            circuits.
  JOURNAL   J Biol Eng. 2019 Mar 1;13:20. doi: 10.1186/s13036-019-0151-x. 
            eCollection 2019.
  PUBMED    30867677
REFERENCE   2  (bases 1 to 2709)
  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: "J Biol 
            Eng."; date: "2019-03-1"; volume: "13:20. doi"; pages: " 
            10.1186/s13036-019-0151-x. eCollection 2019"
COMMENT     Sequence Label: pRC3EGFP
FEATURES             Location/Qualifiers
     source          1..2709
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             71..82
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(89..91,92..94,95..808)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(89..91,92..94,95..805)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(134..155)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(395..414)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     742..763
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      820..906
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(941..958)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1075..1620)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(1155..1174)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1734..1828)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1849..2508)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2423..2442
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2509..2611)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     2677..2695
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagtaac accgtgcgtg ttgactattt tacctctggc ggtgataatg gttgcatgta
       61 cgaattcatt aaagaggaga aaggtaccat ggtgagcaag ggcgaggagc tgttcaccgg
      121 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
      181 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
      241 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
      301 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
      361 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
      421 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
      481 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
      541 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
      601 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
      661 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
      721 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
      781 tctcggcatg gacgagctgt acaagtaatc tagaggcatc aaataaaacg aaaggctcag
      841 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
      901 acaaatccgc cgccctagac ctagggatat attccgcttc ctcgctcact gactcgctac
      961 gctcggtcgt tcgactgcgg cgagcggaaa tggcttacga acggggcgga gatttcctgg
     1021 aagatgccag gaagatactt aacagggaag tgagagggcc gcggcaaagc cgtttttcca
     1081 taggctccgc ccccctgaca agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa
     1141 cccgacagga ctataaagat accaggcgtt tccccctggc ggctccctcg tgcgctctcc
     1201 tgttcctgcc tttcggttta ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc
     1261 cacgcctgac actcagttcc gggtaggcag ttcgctccaa gctggactgt atgcacgaac
     1321 cccccgttca gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     1381 aaagacatgc aaaagcacca ctggcagcag ccactggtaa ttgatttaga ggagttagtc
     1441 ttgaagtcat gcgccggtta aggctaaact gaaaggacaa gttttggtga ctgcgctcct
     1501 ccaagccagt tacctcggtt caaagagttg gtagctcaga gaaccttcga aaaaccgccc
     1561 tgcaaggcgg ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa acgatctcaa
     1621 gaagatcatc ttattaatca gataaaatat ttctagattt cagtgcaatt tatctcttca
     1681 aatgtagcac ctgaagtcag ccccatacga tataagttgt tactagtgct tggattctca
     1741 ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgag
     1801 gtcattactg gatctatcaa caggagtcca agcgagctcg atatcaaatt acgccccgcc
     1861 ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc
     1921 acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata
     1981 atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc
     2041 aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc
     2101 tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag
     2161 aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc
     2221 atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat
     2281 tgccatacgg aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg
     2341 ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac
     2401 ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg
     2461 ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt
     2521 agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg
     2581 gtgaaagttg gaacctctta cgtgccgatc aacgtctcat tttcgccaga tatcgacgtc
     2641 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     2701 cgtcttcac
//
