LOCUS       sequence-436631-        7615 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To track the natural Cra promoter's transcriptional dynamics.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7615)
  AUTHORS   Dash S, Jagadeesan R, Baptista ISC, Chauhan V, Kandavalli V, 
            Oliveira SMD, Ribeiro AS
  TITLE     A library of reporters of the global regulators of gene expression 
            in Escherichia coli.
  JOURNAL   mSystems. 2024 Jun 18;9(6):e0006524. doi: 10.1128/msystems.00065-24.
            Epub 2024 Apr 30.
  PUBMED    38687030
REFERENCE   2  (bases 1 to 7615)
  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: "mSystems.";
            date: "2024-06-18"; volume: "9(6):e0006524. doi"; pages: " 
            10.1128/msystems.00065-24. Epub 2024 Apr 30"
COMMENT     Sequence Label: Cra-GFP
FEATURES             Location/Qualifiers
     source          1..7615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1025..1275
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(1278..2033)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      2124..2343
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     rep_origin      2411..2942
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      2419..3033
                     /label=oriV
                     /note="origin of replication for the bacterial F plasmid"
     promoter        3891..3993
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3994..4653
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4060..4079)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(4788..4882)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4945..5658)
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4985..5005)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     5600..5628
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     RBS             5664..5672
                     /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)"
     terminator      5924..6010
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(6168..7139)
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     CDS             complement(join(7139..7615,1..699))
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
ORIGIN
        1 agtttcaatg gccagtcgga gcatcaggtg tggatcggtg ggcagtttac cttcatcaaa
       61 tttgcccatt aactcagttt caatacggtg cagagccaga caggaaggaa taatgtcaag
      121 ccccggccag caagtgggct ttattgcata agtgacatcg tccttttccc caagatagaa
      181 aggcaggaga gtgtcttctg catgaatatg aagatctggt acccatccgt gatacattga
      241 ggctgttccc tgggggtcgt taccttccac gagcaaaaca cgtagcccct tcagagccag
      301 atcctgagca agatgaacag aaactgaggt tttgtaaacg ccacctttat gggcagcaac
      361 cccgatcacc ggtggaaata cgtcttcagc acgtcgcaat cgcgtaccaa acacatcacg
      421 catatgatta atttgttcaa ttgtataacc aacacgttgc tcaacccgtc ctcgaatttc
      481 catatccggg tgcggtagtc gccctgcttt ctcggcatct ctgatagcct gagaagaaac
      541 cccaactaaa tccgctgctt cacctattct ccagcgccgg gttattttcc tcgcttccgg
      601 gctgtcatca ttaaactgtg caatggcgat agccttcgtc atttcatgac cagcgtttat
      661 gcactggtta agtgtttcca tgagtttcat tctgaacatc ctttaatcat tgctttgcgt
      721 ttttttatta aatcttgcaa tttactgcaa agcaacaaca aaatcgcaaa gtcatcaaaa
      781 aaccgcaaag ttgtttaaaa taagagcaac actacaaaag gagataagaa gagcacatac
      841 ctcagtcact tattatcact agcgctcgcc gcagccgtgt aaccgagcat agcgagcgaa
      901 ctggcgagga agcaaagaag aactgttctg tcagatagct cttacgctca gcgcaagaag
      961 aaatatccac cgtgggaaaa actccaggta gaggtacaca cgcggatagc caattcagag
     1021 taataaactg tgataatcaa ccctcatcaa tgatgacgaa ctaacccccg atatcaagtc
     1081 acatgacgaa gggaaagaga aggaaatcaa ctgtgacaaa ctgccctcaa atttggcttc
     1141 cttaaaaatt acagttcaaa aagtatgaga aaatccatgc aggctgaagg aaacagcaaa
     1201 actgtgacaa attaccctca gtaggtcaga acaaatgtga cgaaccaccc tcaaatctgt
     1261 gacagataac cctcagacta tcctgtcgtc atggaagtga tatcgcggaa ggaaaatacg
     1321 atatgagtcg tctggcggcc tttctttttc tcaatgtatg agaggcgcat tggagttctg
     1381 ctgttgatct cattaacaca gacctgcagg aagcggcggc ggaagtcagg catacgctgg
     1441 taactttgag gcagctggta acgctctatg atccagtcga ttttcagaga gacgatgcct
     1501 gagccatccg gcttacgata ctgacacagg gattcgtata aacgcatggc atacggattg
     1561 gtgatttctt ttgtttcact aagccgaaac tgcgtaaacc ggttctgtaa cccgataaag
     1621 aagggaatga gatatgggtt gatatgtaca ctgtaaagcc ctctggatgg actgtgcgca
     1681 cgtttgataa accaaggaaa agattcatag cctttttcat cgccggcatc ctcttcaggg
     1741 cgataaaaaa ccacttcctt ccccgcgaaa ctcttcaatg cctgccgtat atccttactg
     1801 gcttccgcag aggtcaatcc gaatatttca gcatatttag caacatggat ctcgcagata
     1861 ccgtcatgtt cctgtagggt gccatcagat tttctgatct ggtcaacgaa cagatacagc
     1921 atacgttttt gatcccggga gagactatat gccgcctcag tgaggtcgtt tgactggacg
     1981 attcgcgggc tatttttacg tttcttgtga ttgataaccg ctgtttccgc catgacagat
     2041 ccatgtgaag tgtgacaagt ttttagattg tcacactaaa taaaaaagag tcaataagca
     2101 gggataactt tgtgaaaaaa cagcttcttc tgagggcaat ttgtcacagg gttaagggca
     2161 atttgtcaca gacaggactg tcatttgagg gtgatttgtc acactgaaag ggcaatttgt
     2221 cacaacacct tctctagaac cagcatggat aaaggcctac aaggcgctct aaaaaagaag
     2281 atctaaaaac tataaaaaaa ataattataa aaatatcccc gtggataagt ggataacccc
     2341 aagggaagtt ttttcaggca tcgtgtgtaa gcagaatata taagtgctgt tccctggtgc
     2401 ttcctcgctc actcgaccgg gagggttcga gaaggggggg cacccccctt cggcgtgcgc
     2461 ggtcacgcgc acagggcgca gccctggtta aaaacaaggt ttataaatat tggtttaaaa
     2521 gcaggttaaa agacaggtta gcggtggccg aaaaacgggc ggaaaccctt gcaaatgctg
     2581 gattttctgc ctgtggacag cccctcaaat gtcaataggt gcgcccctca tctgtcagca
     2641 ctctgcccct caagtgtcaa ggatcgcgcc cctcatctgt cagtagtcgc gcccctcaag
     2701 tgtcaatacc gcagggcact tatccccagg cttgtccaca tcatctgtgg gaaactcgcg
     2761 taaaatcagg cgttttcgcc gatttgcgag gctggccagc tccacgtcgc cggccgaaat
     2821 cgagcctgcc cctcatctgt caacgccgcg ccgggtgagt cggcccctca agtgtcaacg
     2881 tccgcccctc atctgtcagt gagggccaag ttttccgcga ggtatccaca acgccggcgg
     2941 ccggccgcgg tgtctcgcac acggcttcga cggcgtttct ggcgcgtttg cagggccata
     3001 gacggccgcc agcccagcgg cgagggcaac cagccgaggg cttcgccctg tcgctcgact
     3061 gcggcgagca ctactggctg taaaaggaca gaccacatca tggttctgtg ttcattaggt
     3121 tgttctgtcc attgctgaca taatccgctc cacttcaacg taacaccgca cgaagatttc
     3181 tattgttcct gaaggcatat tcaaatcgtt ttcgttaccg cttgctggca tcatgacaga
     3241 acactacttc ctataaacgc tacacaggct cctgagatta ataatgcgga tctctacgat
     3301 aatgggagat tttcccgact gtttcgttcg cttctcagtg gataacagcc agcttctctg
     3361 tttaacagac aaaaacagca tatccactca gttccacatt tccatataaa ggccaaggca
     3421 tttattctca ggataattgt ttcagcatcg caaccgcatc agactccggc atcgcaaact
     3481 gcacccggtg ccgggcagcc acatccagcg caaaaacctt cgtgtagact tccgttgaac
     3541 tgatggactt atgtcccatc aggctttgca gaactttcag cggtataccg gcatacagca
     3601 tgtgcatcgc ataggaatgg cggaacgtat gtggtgtgac cggaacagag aacgtcacac
     3661 cgtcagcagc agcggcggca accgcctccc caatccaggt cctgaccgtt ctgtccgtca
     3721 cttcccagat ccgcgctttc tctgtccttc ctgtgcgacg gttacgccgc tccatgagct
     3781 tatcgcgaat aaatacctgt gacggaagat cacttcgcag aataaataaa tcctggtgtc
     3841 cctgttgata ccgggaagcc ctgggccaac ttttggcgaa aatgagacgt tgatcggcac
     3901 gtaagaggtt ccaactttca ccataatgaa ataagatcac taccgggcgt attttttgag
     3961 ttatcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc
     4021 accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct
     4081 caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag
     4141 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct
     4201 catccggaat ttcgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac
     4261 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac
     4321 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa
     4381 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc
     4441 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc
     4501 gttttcacga tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt
     4561 caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa
     4621 cagtactgcg atgagtggca gggcggggcg taattttttt aaggcagtta ttggtgccct
     4681 taaacgcctg gttgctacgc ctgaataagt gataataagc ggatgaatgg cagaaattcg
     4741 atgataagct gtcaaacatg agaattggtc tcgacgagta tgcttggatt ctcaccaata
     4801 aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgaggtcatt
     4861 actggatcta tcaacaggag tccaagcgac gtctgtgcaa gtactactgt tctgcagtca
     4921 cttgaattcg atacccagct gttatttgta tagttcatcc atgccatgtg taatcccagc
     4981 agcggttaca aactcaagaa ggaccatgtg gtctctcttt tcgttgggat ctttcgaaag
     5041 ggcagattgt gtggacaggt aatggttgtc tggtaaaagg acagggccat cgccaatcgg
     5101 agtattttgt tgataatggt ctgctagttg aacgcttcca tcttcaatgt tgtgtctaat
     5161 tttgaagtta actttgattc cattcttttg tttgtctgcc atgatgtata cattgtgtga
     5221 gttatagttg tattccaatt tgtgtccaag aatgtttcca tcttctttaa aatcaatacc
     5281 ttttaactcg attctattaa caagggtatc accttcaaac ttgacttcag cacgtgtctt
     5341 gtagttcccg tcatctttga aaaatatagt tctttcctgt acgtaacctt cgggcatggc
     5401 actcttgaaa aagtcatgct gtttcatgtg atctgggtat ctcgcaaagc attgaacacc
     5461 ataaccgaaa gtagtgacaa gtgttggcca cggaacaggt agttttccag tagtgcaaat
     5521 aaatttaagg gtaagttttc cgtatgttgc atcaccttca ccctctccac tgacagaaaa
     5581 tttgtgccca ttaacatcac catctaattc aacaagaatt gggacaactc cagtgaaaag
     5641 ttcttctcct ttacgcatat tatacctcct taatttagtg tcatctacaa ctggcataat
     5701 tgccccttgc gtaaaatgta aaaaccatag ccattgtact ggtatgggtt aaattcatta
     5761 acatctaagc gcagaaaatt aactgcggca actgctttta ttccaggttc ccactgattt
     5821 cgcaaaaaaa gcccaacgtg acatgttggg ctgtaaattg cgcattgaga tcattcccta
     5881 gggaccgtct cgagagaatc aatattaatc caacgcgtgg catcaaataa aacgaaaggc
     5941 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     6001 taggacaaat ccgccgccct agacttaggc gttcggctgc ggcgagcggt atcagctcac
     6061 tcaaagaagt ggggcccaga gagaatattc aggccagtta tgctttctgg cctgtaacaa
     6121 aggacattaa gtaaagacag ataaacgtag actaaaacgt ggtcgcatca gggtgctggc
     6181 ttttcaagtt ccttaagaat ggcctcaatt ttctctatac actcagttgg aacacgggac
     6241 ctgtccaggt taagcaccat tttatcgccc ttatacaata ctgtcgctcc aggagcaaac
     6301 tgatgtcgtg agcttaaact agttcttgat gcagatgacg ttttaagcac agaagttaaa
     6361 agagtgataa cttcttcagc ttcaaatatc accccagctt ttttctgctc atgaaggtta
     6421 gatgcctgct gcttaagtaa ttcctcttta tctgtaaagg ctttttgaag tgcatcacct
     6481 gaccgggcag atagttcacc ggggtgagaa aaaagagcaa caactgattt aggcaatttg
     6541 gcggtgttga tacagcgggt aataatctta cgtgaaatat tttccgcatc agccagcgca
     6601 gaaatatttc cagcaaattc attctgcaat cggcttgcat aacgctgacc acgttcataa
     6661 gcacttgttg ggcgataatc gttacccaat ctggataatg cagccatctg ctcatcatcc
     6721 agctcgccaa ccagaacacg ataatcactt tcggtaagtg cagcagcttt acgacggcga
     6781 ctcccatcgg caatttctat gacaccagat actcttcgac cgaacgccgg tgtctgttga
     6841 ccagtcagta gaaaagaagg gatgagatca tccagtgcgt cctcagtaag cagctcctgg
     6901 tcacgttcat tacctgacca tacccgagag gtcttctcaa cactatcacc ccggagcact
     6961 tcaagagtaa acttcacatc ccgaccacat acaggcaaag taatggcatt accgcgagcc
     7021 attactccta cgcgcgcaat taacgaatcc accatcgggg cagctggtgt cgataacgaa
     7081 gtatcttcaa ccggttgagt attgagcgta tgttttggaa taacaggcgc acgcttcatt
     7141 atctaatctc ccagcgtggt ttaatcagac gatcgaaaat ttcattgcag acaggttccc
     7201 aaatagaaag agcatttctc caggcaccag ttgaagagcg ttgatcaatg gcctgttcaa
     7261 aaacagttct catccggatc tgacctttac caacttcatc cgtttcacgt acaacatttt
     7321 ttagaaccat gcttccccag gcatcccgaa tttgctcctc catccacggg gactgagagc
     7381 cattactatt gctgtatttg gtaagcaaaa tacgtacatc aggctcgaac cctttaagat
     7441 caacgttctt gagcagatca cgaagcatat cgaaaaactg cagtgcggag gtgtagtcaa
     7501 acaactcagc aggcgtggga acaatcagca catcagcagc acatacgaca ttaatcgtgc
     7561 cgatacccag gttaggcgcg ctgtcaataa ctatgacatc atagtcatga gcaac
//
