LOCUS       sequence-389829-        4536 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cassette 1: Expresses EGFP under the control of PR promoter, 
            Cassette 2: Expresses frame-shifted CI under the control of PLTetO-1
            promoter, pUC origin of replication, Ampicillin selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4536)
  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 4536)
  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: pRA1SEGFPTcIfm
FEATURES             Location/Qualifiers
     source          1..4536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             76..87
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(94..96,97..99,100..813)
                     /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(94..96,97..99,100..810)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(139..160)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(400..419)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     747..768
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      825..911
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(1012..1600)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1092..1111)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1918..2012)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(2042..2833,2834..2902))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2665..2684
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2903..3007)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3075..3093
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3357..3443)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3455..4162)
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="TKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQSG
                     VGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEYPV
                     FSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPDGM
                     LILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSVVG
                     KVIASQWPEETFG"
     RBS             4174..4185
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(join(4195..4220,4221..4226,4227..4243,4244..4249,
                     4250..4268))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    4225..4243
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4250..4268
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 ctctgctcga gtaacaccgt gcgtgttgac tattttacct ctggcggtga taatggttgc
       61 atgtacgaat tcattaaaga ggagaaaggt accatggtga gcaagggcga ggagctgttc
      121 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
      181 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
      241 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
      301 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
      361 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
      421 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
      481 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
      541 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
      601 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
      661 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
      721 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
      781 atcactctcg gcatggacga gctgtacaag taatctagag gcatcaaata aaacgaaagg
      841 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga
      901 gtaggacaaa tccgccgccc tagacctagt aggggataac gcaggaaaga acatgtgagc
      961 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     1021 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     1081 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     1141 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     1201 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     1261 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     1321 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     1381 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     1441 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     1501 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     1561 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     1621 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     1681 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     1741 aagtatatat gagtaacctg aggctatggc agggcctgcc gccccgacgt tggctgcgag
     1801 ccctgggcct tcacccgaac ttggggggtg gggtggggaa aaggaagaaa cgcgggcgta
     1861 ttggccccaa tggggtctcg gtggggtatc gacagagtgc cagcccctag tgcttggatt
     1921 ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc
     1981 tgaggtcatt actggatcta tcaacaggag tccaagcgag ctcgtaaact tggtctgaca
     2041 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     2101 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     2161 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     2221 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     2281 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     2341 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     2401 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     2461 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     2521 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     2581 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     2641 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     2701 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     2761 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     2821 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     2881 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     2941 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     3001 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     3061 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcacttg ttttggagca
     3121 cggaaagacg atgacggaaa aagagatcgt ggattacgtc gccagtcaag taacaaccgc
     3181 caaaaagttg cgcggaggag ttgtgtttgt ggacgaagta ccgaaaggtc ttaccggaaa
     3241 actcgacgca agaaaaatca gagagatcct cataaaggcc aagaaggatc cattaccgcc
     3301 tttgagtgag ctgataccgc tcgccgcagc cgaacgccct aggtctaggg cggcggattt
     3361 gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc
     3421 gactgagcct ttcgttttat ttgatgcctc tagatcagcc aaacgtctct tcaggccact
     3481 gactagcgat aactttcccc acaacggaac aactctcatt gcatgggatc attgggtact
     3541 gtgggtttag tggttgtaaa aacacctgac cgctatccct gatcagtttc ttgaaggtaa
     3601 actcatcacc cccaagtctg gctatgcaga aatcacctgg ctcaacagcc tgctcagggt
     3661 caacgagaat taacattccg tcaggaaagc ttggcttgga gcctgttggt gcggtcatgg
     3721 aattaccttc aacctcaagc cagaatgcag aatcactggc ttttttggtt gtgcttaccc
     3781 atctctccgc atcacctttg gtaaaggttc taagctcagg tgagaacatc cctgcctgaa
     3841 catgagaaaa aacagggtac tcatactcac ttctaagtga cggctgcata ctaaccgctt
     3901 catacatctc gtagatttct ctggcgattg aagggctaaa ttcttcaacg ctaactttga
     3961 gaatttttgc aagcaatgcg gcgttataag catttaatgc attgatgcca ttaaataaag
     4021 caccaacgcc tgactgcccc atccccatct tgtctgcgac agattcctgg gataagccaa
     4081 gttcattttt ctttttttca taaattgctt taaggcgacg tgcgtcctca agctgctctt
     4141 gtgttaatgg tttctttttt gtgccatggt acctttctcc tctttaatga attcggtcag
     4201 tgcgtcctgc tgatgtgctc agtatctcta tcactgatag ggatgtcaat ctctatcact
     4261 gatagggact cgaggtgaag acgaactgca gcagattctc gcatgccaga gatcctattt
     4321 ttggcaatca aatcattccg gatactgcga ttttaagtgt tgttccattc catcacggtt
     4381 ttggaatgtt tactacactc ggatatttga tatgtggatt tcgagtcgtc ttaatgtata
     4441 gatttgaaga agagctgttt ttacgatccc ttcaggatta caaaattcaa agtgcgttgc
     4501 tagtaccaac cctattttca ttcttcgcca aaagca
//
