LOCUS       sequence-448715-        7868 bp    DNA     circular SYN 15-APR-2025
DEFINITION  LacO3O1 and tetA promoters in tandem conformation with 250bp 
            distance between them, expressing mCherry with pBELO_L6-PT3-T4 
            backbone and chloramphenicol resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7868)
  AUTHORS   Chauhan V, Baptista ISC, Arsh AM, Jagadeesan R, Dash S, Ribeiro AS
  TITLE     Transcription Attenuation in Synthetic Promoters in Nonoverlapping 
            Tandem Formation.
  JOURNAL   Biochemistry. 2024 Jul 12. doi: 10.1021/acs.biochem.4c00012.
  PUBMED    38997112
REFERENCE   2  (bases 1 to 7868)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Biochemistry. 2024 Jul 12. doi: 10.1021/acs.biochem.4c00012."
COMMENT     Sequence Label: PLacO3O1-PtetA 250 bp with pBELO_L6-PT3-T4_CAM...
FEATURES             Location/Qualifiers
     source          1..7868
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(102..1073)
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPTAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     CDS             complement(1073..2248)
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     misc_feature    2574..2824
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(2827..3582)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      3673..3892
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     rep_origin      3960..4491
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      3968..4582
                     /label=oriV
                     /note="origin of replication for the bacterial F plasmid"
     promoter        5440..5542
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             5543..6202
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5609..5628)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(6337..6431)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     protein_bind    6568..6589
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6604..6609,6610..6627,6628..6634)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6642..6658
                     /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)."
     primer_bind     6647..6669
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        6849..6904
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    6855..6873
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    6885..6903
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             6942..6950
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             6956..7666
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(7101..7119)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7369..7388
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      7726..7812
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aagtggggcc cagagagaat attcaggcca gttatgcttt ctggcctgta acaaaggaca
       61 ttaagtaaag acagataaac gtagactaaa acgtggtcgc atcagggtgc tggcttttca
      121 agttccttaa gaatggcctc aattttctct atacactcag ttggaacacg ggacctgtcc
      181 aggttaagca ccattttatc gcccttatac aatactgtcg ctccaggagc aaactgatgt
      241 cgtgagctta aactagttct tgatgcagat gacgttttaa gcacagaagt taaaagagtg
      301 ataacttctt cagcttcaaa tatcacccca gcttttttct gctcatgaag gttagatgcc
      361 tgctgcttaa gtaattcctc tttatctgta aaggcttttt gaagtgcatc acctgaccgg
      421 gcagatagtt caccggggtg agaaaaaaga gcaacaactg atttaggcaa tttggcggtg
      481 ttgatacagc gggtaataat cttacgtgaa atattttccg catcagccag cgcagaaata
      541 tttccagcaa attcattctg caatcggctt gcataacgct gaccacgttc ataagcactt
      601 gttgggcgat aatcgttacc caatctggat aatgcagcca tctgctcatc atccagctcg
      661 ccaaccagaa cacgataatc actttcggta agtgcagcag ctttacgacg gcgactccca
      721 tcggcaattt ctatgacacc agatactctt cgaccgaacg ccggtgtctg ttgaccagtc
      781 agtagaaaag aagggatgag atcatccagt gcgtcctcag taagcagctc ctggtcacgt
      841 tcattacctg accatacccg agaggtcttc tcaacactat caccccggag cacttcaaga
      901 gtaaacttca catcccgacc acatacaggc aaagtaatgg cattaccgcg agccattact
      961 cctacgcgcg caattaacga atccaccatc ggggcagttg gtgtcgataa cgaagtatct
     1021 tcaaccggtt gagtattgag cgtatgtttt ggaataacag gcgcacgctt cattatctaa
     1081 tctcccagcg tggtttaatc agacgatcga aaatttcatt gcagacaggt tcccaaatag
     1141 aaagagcatt tctccaggca ccagttgaag agcgttgatc aatggcctgt tcaaaaacag
     1201 ttctcatccg gatctgacct ttaccaactt catccgtttc acgtacaaca ttttttagaa
     1261 ccatgcttcc ccaggcatcc cgaatttgct cctccatcca cggggactga gagccattac
     1321 tattgctgta tttggtaagc aaaatacgta catcaggctc gaacccttta agatcaacgt
     1381 tcttgagcag atcacgaagc atatcgaaaa actgcagtgc ggaggtgtag tcaaacaact
     1441 cagcaggcgt gggaacaatc agcacatcag cagcacatac gacattaatc gtgccgatac
     1501 ccaggttagg cgcgctgtca ataactatga catcatagtc atgagcaaca gtttcaatgg
     1561 ccagtcggag catcaggtgt ggatcggtgg gcagtttacc ttcatcaaat ttgcccatta
     1621 actcagtttc aatacggtgc agagccagac aggaaggaat aatgtcaagc cccggccagc
     1681 aagtgggctt tattgcataa gtgacatcgt ccttttcccc aagatagaaa ggcaggagag
     1741 tgtcttctgc atgaatatga agatctggta cccatccgtg atacattgag gctgttccct
     1801 gggggtcgtt accttccacg agcaaaacac gtagcccctt cagagccaga tcctgagcaa
     1861 gatgaacaga aactgaggtt ttgtaaacgc cacctttatg ggcagcaacc ccgatcaccg
     1921 gtggaaatac gtcttcagca cgtcgcaatc gcgtaccaaa cacatcacgc atatgattaa
     1981 tttgttcaat tgtataacca acacgttgct caacccgtcc tcgaatttcc atatccgggt
     2041 gcggtagtcg ccctgctttc tcggcatctc tgatagcctg agaagaaacc ccaactaaat
     2101 ccgctgcttc acctattctc cagcgccggg ttattttcct cgcttccggg ctgtcatcat
     2161 taaactgtgc aatggcgata gccttcgtca tttcatgacc agcgtttatg cactggttaa
     2221 gtgtttccat gagtttcatt ctgaacatcc tttaatcatt gctttgcgtt tttttattaa
     2281 atcttgcaat ttactgcaaa gcaacaacaa aatcgcaaag tcatcaaaaa accgcaaagt
     2341 tgtttaaaat aagagcaaca ctacaaaagg agataagaag agcacatacc tcagtcactt
     2401 attatcacta gcgctcgccg cagccgtgta accgagcata gcgagcgaac tggcgaggaa
     2461 gcaaagaaga actgttctgt cagatagctc ttacgctcag cgcaagaaga aatatccacc
     2521 gtgggaaaaa ctccaggtag aggtacacac gcggatagcc aattcagagt aataaactgt
     2581 gataatcaac cctcatcaat gatgacgaac taacccccga tatcaagtca catgacgaag
     2641 ggaaagagaa ggaaatcaac tgtgacaaac tgccctcaaa tttggcttcc ttaaaaatta
     2701 cagttcaaaa agtatgagaa aatccatgca ggctgaagga aacagcaaaa ctgtgacaaa
     2761 ttaccctcag taggtcagaa caaatgtgac gaaccaccct caaatctgtg acagataacc
     2821 ctcagactat cctgtcgtca tggaagtgat atcgcggaag gaaaatacga tatgagtcgt
     2881 ctggcggcct ttctttttct caatgtatga gaggcgcatt ggagttctgc tgttgatctc
     2941 attaacacag acctgcagga agcggcggcg gaagtcaggc atacgctggt aactttgagg
     3001 cagctggtaa cgctctatga tccagtcgat tttcagagag acgatgcctg agccatccgg
     3061 cttacgatac tgacacaggg attcgtataa acgcatggca tacggattgg tgatttcttt
     3121 tgtttcacta agccgaaact gcgtaaaccg gttctgtaac ccgataaaga agggaatgag
     3181 atatgggttg atatgtacac tgtaaagccc tctggatgga ctgtgcgcac gtttgataaa
     3241 ccaaggaaaa gattcatagc ctttttcatc gccggcatcc tcttcagggc gataaaaaac
     3301 cacttccttc cccgcgaaac tcttcaatgc ctgccgtata tccttactgg cttccgcaga
     3361 ggtcaatccg aatatttcag catatttagc aacatggatc tcgcagatac cgtcatgttc
     3421 ctgtagggtg ccatcagatt ttctgatctg gtcaacgaac agatacagca tacgtttttg
     3481 atcccgggag agactatatg ccgcctcagt gaggtcgttt gactggacga ttcgcgggct
     3541 atttttacgt ttcttgtgat tgataaccgc tgtttccgcc atgacagatc catgtgaagt
     3601 gtgacaagtt tttagattgt cacactaaat aaaaaagagt caataagcag ggataacttt
     3661 gtgaaaaaac agcttcttct gagggcaatt tgtcacaggg ttaagggcaa tttgtcacag
     3721 acaggactgt catttgaggg tgatttgtca cactgaaagg gcaatttgtc acaacacctt
     3781 ctctagaacc agcatggata aaggcctaca aggcgctcta aaaaagaaga tctaaaaact
     3841 ataaaaaaaa taattataaa aatatccccg tggataagtg gataacccca agggaagttt
     3901 tttcaggcat cgtgtgtaag cagaatatat aagtgctgtt ccctggtgct tcctcgctca
     3961 ctcgaccggg agggttcgag aagggggggc accccccttc ggcgtgcgcg gtcacgcgca
     4021 cagggcgcag ccctggttaa aaacaaggtt tataaatatt ggtttaaaag caggttaaaa
     4081 gacaggttag cggtggccga aaaacgggcg gaaacccttg caaatgctgg attttctgcc
     4141 tgtggacagc ccctcaaatg tcaataggtg cgcccctcat ctgtcagcac tctgcccctc
     4201 aagtgtcaag gatcgcgccc ctcatctgtc agtagtcgcg cccctcaagt gtcaataccg
     4261 cagggcactt atccccaggc ttgtccacat catctgtggg aaactcgcgt aaaatcaggc
     4321 gttttcgccg atttgcgagg ctggccagct ccacgtcgcc ggccgaaatc gagcctgccc
     4381 ctcatctgtc aacgccgcgc cgggtgagtc ggcccctcaa gtgtcaacgt ccgcccctca
     4441 tctgtcagtg agggccaagt tttccgcgag gtatccacaa cgccggcggc cggccgcggt
     4501 gtctcgcaca cggcttcgac ggcgtttctg gcgcgtttgc agggccatag acggccgcca
     4561 gcccagcggc gagggcaacc agccgagggc ttcgccctgt cgctcgactg cggcgagcac
     4621 tactggctgt aaaaggacag accacatcat ggttctgtgt tcattaggtt gttctgtcca
     4681 ttgctgacat aatccgctcc acttcaacgt aacaccgcac gaagatttct attgttcctg
     4741 aaggcatatt caaatcgttt tcgttaccgc ttgctggcat catgacagaa cactacttcc
     4801 tataaacgct acacaggctc ctgagattaa taatgcggat ctctacgata atgggagatt
     4861 ttcccgactg tttcgttcgc ttctcagtgg ataacagcca gcttctctgt ttaacagaca
     4921 aaaacagcat atccactcag ttccacattt ccatataaag gccaaggcat ttattctcag
     4981 gataattgtt tcagcatcgc aaccgcatca gactccggca tcgcaaactg cacccggtgc
     5041 cgggcagcca catccagcgc aaaaaccttc gtgtagactt ccgttgaact gatggactta
     5101 tgtcccatca ggctttgcag aactttcagc ggtataccgg catacagcat gtgcatcgca
     5161 taggaatggc ggaacgtatg tggtgtgacc ggaacagaga acgtcacacc gtcagcagca
     5221 gcggcggcaa ccgcctcccc aatccaggtc ctgaccgttc tgtccgtcac ttcccagatc
     5281 cgcgctttct ctgtccttcc tgtgcgacgg ttacgccgct ccatgagctt atcgcgaata
     5341 aatacctgtg acggaagatc acttcgcaga ataaataaat cctggtgtcc ctgttgatac
     5401 cgggaagccc tgggccaact tttggcgaaa atgagacgtt gatcggcacg taagaggttc
     5461 caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt
     5521 ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat
     5581 atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta
     5641 taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca
     5701 caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt
     5761 tcgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac
     5821 cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt
     5881 ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta
     5941 tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt
     6001 caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcacgat
     6061 gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca
     6121 tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga
     6181 tgagtggcag ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg
     6241 ttgctacgcc tgaataagtg ataataagcg gatgaatggc agaaattcga tgataagctg
     6301 tcaaacatga gaattggtct cgacgagtat gcttggattc tcaccaataa aaaacgcccg
     6361 gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta ctggatctat
     6421 caacaggagt ccaagcgacg tctgtgcaag tactactgtt ctgcagtcac ttgaattcga
     6481 tacccagctg ttatttgtat agttcatcca tgccatgtgt aatcccagca gcggttactg
     6541 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     6601 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     6661 tcacacagga gatacgaatt tacgtataga gcgggtcatc gaaaggttat actctcgtag
     6721 ttaacatcta gcccggccct atcagtacag cagtgccttg aatgacatac tcatcattaa
     6781 attttctcta cagccaaacg accaagtgca tttccaggga gcgcgacgac gagcagtcat
     6841 gaatttttgt tgacactcta tcattgatag agttatttta ccactcccta tcagtgatag
     6901 agaaaagtga aatgttcaca ccaattgata atatacaaga ttaaggaggt aaagaatggt
     6961 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggttca
     7021 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     7081 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     7141 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     7201 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     7261 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aagacggcga
     7321 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     7381 gaagaagact atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgcgct
     7441 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     7501 caagaccacc tacaaggcca agaagcccgt gcaactgccc ggcgcgtaca acgtcaacat
     7561 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     7621 cgagggccgc cactccaccg gcggcatgga cgagctgtat aagtaagata tctatcgccc
     7681 tagggaccgt ctcgagagaa tcaatattaa tccaacgcgt ggcatcaaat aaaacgaaag
     7741 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     7801 agtaggacaa atccgccgcc ctagacttag gcgttcggct gcggcgagcg gtatcagctc
     7861 actcaaag
//
