LOCUS       sequence-356602-        3760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aTc-inducible expression of mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3760)
  AUTHORS   Butcher RJ, Tabor JJ
  TITLE     Real-time detection of response regulator phosphorylation dynamics 
            in live bacteria.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Aug 30;119(35):e2201204119. doi: 
            10.1073/pnas.2201204119. Epub 2022 Aug 22.
  PUBMED    35994658
REFERENCE   2  (bases 1 to 3760)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2022-08-30"; volume: "119(35):e2201204119. 
            doi"; pages: " 10.1073/pnas.2201204119. Epub 2022 Aug 22"
COMMENT     Sequence Label: pRB035
FEATURES             Location/Qualifiers
     source          1..3760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(20..608)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(100..119)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(696..790)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(811..1470)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAKCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1385..1404
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     1639..1657
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1677..1695,1696..1701,1702..1718,1719..1724,
                     1725..1750)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1677..1695
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1702..1720
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      2555..2626
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2642..2669
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2828..2862
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     CDS             2897..3523
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(2973..2992)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      3529..3615
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
       61 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
      121 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
      181 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
      241 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
      301 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
      361 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
      421 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
      481 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
      541 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
      601 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
      661 cacgttaagg gattttggtc atgactagtg cttggattct caccaataaa aaacgcccgg
      721 cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc
      781 aacaggagtc caagcgagct cgatatcaaa ttacgccccg ccctgccact catcgcagta
      841 ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac
      901 ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa
      961 aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac
     1021 ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag
     1081 gttttcacca taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc
     1141 gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca
     1201 agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac gaaattccgg
     1261 atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt
     1321 tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca
     1381 tttagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac
     1441 ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga
     1501 taactcaaaa aatacgaccg gtagtgatct tatttcatta tggtgaaagt tggaacctct
     1561 tacgggccgg ccaacgtctc attttcgcca gatatcgacg tctaagaaac cattattatc
     1621 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca cctcgatccc
     1681 tatcagtgat agagattgac atccctatca gtgatagaga tactgagcac atcagcagga
     1741 cgcactgacc gggcccaagt tcacttaaaa aggagatcaa caatgaaagc aattttcgta
     1801 ctgaaacatc ttaatcatgc ctaggaagtt ttctaatggt ttcgaaaggt gaggaggata
     1861 acatggcgtc gctgcctgct acacatgaac tgcatatttt cggcagcatc aacggagttg
     1921 atttcgacat ggtgggccaa ggcaccggca acccgaacga cggctatgaa gaattgaatt
     1981 tgaaatctac caagggcgac ttgcaattta gtccgtggat tctggtaccg catattggtt
     2041 atggttttca tcagtatctg ccctacccag acggaatgag cccatttcag gcggctatgg
     2101 tggatggcag tggctatcag gtccatcgca ccatgcaatt cgaggatggg gcgagcctga
     2161 ccgtgaatta tcggtacact tacgaaggtt cacatattaa aggtgaagca caagttaaag
     2221 gcaccggctt cccggcggat ggcccagtga tgaccaacag cctgacggcg gcggactggt
     2281 gtcgtagcaa aaagacgtat cccaatgata aaacaattat ttctaccttt aaatggagtt
     2341 acaccacggg taacggtaaa cgctaccgta gcacggctcg caccacatac acctttgcga
     2401 aacccatggc cgcgaactat ctgaaaaatc agccgatgta tgtgtttcgc aaaacggaac
     2461 tgaaacattc gaaaactgaa ctcaatttca aagagtggca aaaagccttc accgacgtaa
     2521 tgggcatgga tgaactgtac aaatgaccag gcatcaaata aaacgaaagg ctcagtcgaa
     2581 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac
     2641 tggctcacct tcgggtgggc ctttctgcgt ttatagtaaa tcactgcata attcgtgtcg
     2701 ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa cggttctggc
     2761 aaatattctg aaatgagctg ctcgtacccg tggatcctct agagtcgacc tgcaggcatg
     2821 caagcttttt acggctagct cagtcctagg tatagtgcta gcccagccag agaaacactc
     2881 tttaacaggg ggtagtatga tgtctcgttt agataaaagt aaagtgatta acagcgcatt
     2941 agagctgctt aatgaggtcg gaatcgaagg tttaacaacc cgtaaactcg cccagaagct
     3001 aggtgtagag cagcctacat tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc
     3061 cttagccatt gagatgttag ataggcacca tactcacttt tgccctttag aaggggaaag
     3121 ctggcaagat tttttacgta ataacgctaa aagttttaga tgtgctttac taagtcatcg
     3181 cgatggagca aaagtacatt taggtacacg gcctacagaa aaacagtatg aaactctcga
     3241 aaatcaatta gcctttttat gccaacaagg tttttcacta gagaatgcat tatatgcact
     3301 cagcgcagtg gggcatttta ctttaggttg cgtattggaa gatcaagagc atcaagtcgc
     3361 taaagaagaa agggaaacac ctactactga tagtatgccg ccattattac gacaagctat
     3421 cgaattattt gatcaccaag gtgcagagcc agccttctta ttcggccttg aattgatcat
     3481 atgcggatta gaaaaacaac ttaaatgtga aagtgggtct taaggcatca aataaaacga
     3541 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     3601 ctgagtagga caaatccgcc gccctagacc tagggcgttc ggctgcggcg agcggtatca
     3661 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc atgcaagaac
     3721 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
//
