LOCUS       sequence-390100-        4382 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cassette 1: Expresses frame-shifted CI under PLLacO-1 promoter, 
            Cassette 2: Expresses frame-shifted CI under PLTetO-1 promoter, 
            ColE1 origin of replication, Ampicillin selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4382)
  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 4382)
  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: pLA2ScIfmTcIfm
FEATURES             Location/Qualifiers
     source          1..4382
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    9..25
                     /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)."
     protein_bind    32..48
                     /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)."
     RBS             90..101
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             113..820
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="TKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQSG
                     VGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEYPV
                     FSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPDGM
                     LILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSVVG
                     KVIASQWPEETFG"
     terminator      832..918
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(1083..1671)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1163..1182)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1759..1853)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(1883..2674,2675..2743))
                     /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     2506..2525
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2744..2848)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2916..2934
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3198..3284)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3296..4003)
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="TKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQSG
                     VGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEYPV
                     FSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPDGM
                     LILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSVVG
                     KVIASQWPEETFG"
     RBS             4015..4026
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(join(4036..4061,4062..4067,4068..4084,4085..4090,
                     4091..4109))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    4066..4084
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4091..4109
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 ctcgagaatt gtgagcggat aacaattgac attgtgagcg gataacaaga tactgagcac
       61 atcagcagga cgcactgacc gaattcatta aagaggagaa aggtaccatg gcacaaaaaa
      121 gaaaccatta acacaagagc agcttgagga cgcacgtcgc cttaaagcaa tttatgaaaa
      181 aaagaaaaat gaacttggct tatcccagga atctgtcgca gacaagatgg ggatggggca
      241 gtcaggcgtt ggtgctttat ttaatggcat caatgcatta aatgcttata acgccgcatt
      301 gcttgcaaaa attctcaaag ttagcgttga agaatttagc ccttcaatcg ccagagaaat
      361 ctacgagatg tatgaagcgg ttagtatgca gccgtcactt agaagtgagt atgagtaccc
      421 tgttttttct catgttcagg cagggatgtt ctcacctgag cttagaacct ttaccaaagg
      481 tgatgcggag agatgggtaa gcacaaccaa aaaagccagt gattctgcat tctggcttga
      541 ggttgaaggt aattccatga ccgcaccaac aggctccaag ccaagctttc ctgacggaat
      601 gttaattctc gttgaccctg agcaggctgt tgagccaggt gatttctgca tagccagact
      661 tgggggtgat gagtttacct tcaagaaact gatcagggat agcggtcagg tgtttttaca
      721 accactaaac ccacagtacc caatgatccc atgcaatgag agttgttccg ttgtggggaa
      781 agttatcgct agtcagtggc ctgaagagac gtttggctga tctagaggca tcaaataaaa
      841 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
      901 ctcctgagta ggacaaatcc gccgccctag acctagggcg ttcggctgcg gcgagcggta
      961 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1021 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1081 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1141 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1201 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1261 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1321 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1381 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     1441 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     1501 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     1561 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     1621 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     1681 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     1741 gtcatgacta gtgcttggat tctcaccaat aaaaaacgcc cggcggcaac cgagcgttct
     1801 gaacaaatcc agatggagtt ctgaggtcat tactggatct atcaacagga gtccaagcga
     1861 gctcgtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     1921 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     1981 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     2041 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     2101 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     2161 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     2221 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     2281 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     2341 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     2401 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     2461 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     2521 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     2581 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     2641 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     2701 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     2761 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     2821 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     2881 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     2941 gtcttcactt gttttggagc acggaaagac gatgacggaa aaagagatcg tggattacgt
     3001 cgccagtcaa gtaacaaccg ccaaaaagtt gcgcggagga gttgtgtttg tggacgaagt
     3061 accgaaaggt cttaccggaa aactcgacgc aagaaaaatc agagagatcc tcataaaggc
     3121 caagaaggat ccattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgccc
     3181 taggtctagg gcggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata
     3241 aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct ctagatcagc
     3301 caaacgtctc ttcaggccac tgactagcga taactttccc cacaacggaa caactctcat
     3361 tgcatgggat cattgggtac tgtgggttta gtggttgtaa aaacacctga ccgctatccc
     3421 tgatcagttt cttgaaggta aactcatcac ccccaagtct ggctatgcag aaatcacctg
     3481 gctcaacagc ctgctcaggg tcaacgagaa ttaacattcc gtcaggaaag cttggcttgg
     3541 agcctgttgg tgcggtcatg gaattacctt caacctcaag ccagaatgca gaatcactgg
     3601 cttttttggt tgtgcttacc catctctccg catcaccttt ggtaaaggtt ctaagctcag
     3661 gtgagaacat ccctgcctga acatgagaaa aaacagggta ctcatactca cttctaagtg
     3721 acggctgcat actaaccgct tcatacatct cgtagatttc tctggcgatt gaagggctaa
     3781 attcttcaac gctaactttg agaatttttg caagcaatgc ggcgttataa gcatttaatg
     3841 cattgatgcc attaaataaa gcaccaacgc ctgactgccc catccccatc ttgtctgcga
     3901 cagattcctg ggataagcca agttcatttt tctttttttc ataaattgct ttaaggcgac
     3961 gtgcgtcctc aagctgctct tgtgttaatg gtttcttttt tgtgccatgg tacctttctc
     4021 ctctttaatg aattcggtca gtgcgtcctg ctgatgtgct cagtatctct atcactgata
     4081 gggatgtcaa tctctatcac tgatagggac tcgaggtgaa gacgaactgc agcagattct
     4141 cgcatgccag agatcctatt tttggcaatc aaatcattcc ggatactgcg attttaagtg
     4201 ttgttccatt ccatcacggt tttggaatgt ttactacact cggatatttg atatgtggat
     4261 ttcgagtcgt cttaatgtat agatttgaag aagagctgtt tttacgatcc cttcaggatt
     4321 acaaaattca aagtgcgttg ctagtaccaa ccctattttc attcttcgcc aaaagcactc
     4381 tg
//
