LOCUS       sequence-426584-        5732 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Always-on expression of mWasabi reporter with transcription 
            activation by wildtype CI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5732)
  AUTHORS   Xiong LL, Garrett MA, Buss MT, Kornfield JA, Shapiro MG
  TITLE     Tunable Temperature-Sensitive Transcriptional Activation Based on 
            Lambda Repressor.
  JOURNAL   ACS Synth Biol. 2022 Jul 15;11(7):2518-2522. doi: 
            10.1021/acssynbio.2c00093. Epub 2022 Jun 15.
  PUBMED    35708251
REFERENCE   2  (bases 1 to 5732)
  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"; doi: 
            "10.1021/acssynbio.2c00093"; journalName: "ACS Synth Biol"; date: 
            "2022-07-15- 15"; volume: "11"; issue: "7"; pages: "2518-2522"
COMMENT     Sequence Label: pCIwt-PRM-Wasabi
FEATURES             Location/Qualifiers
     source          1..5732
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     1984..2003
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     RBS             2160..2171
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2181..2198
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2211..2918
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /label=mWasabi
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYK"
     protein_bind    2998..3012
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     promoter        3125..3201
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3223..3936
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     CDS             3949..3993
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4031..4049)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4045..4092
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4129..4584
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4216..4235)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4426..4447
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4699..5490,5491..5559))
                     /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     5322..5341
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5560..5652)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgctagtc atgccccgcg
     1681 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     1741 cctaatgagt gagctaactt acattaattg cgttgcgccg ggatctcgac gctctccctt
     1801 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     1861 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     1921 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     1981 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     2041 cacgatgcgt ccggcgtaga ggatcgagat tatcaccgcc agaggtaaaa tagtcaacac
     2101 gcacggtgtt agatatttat aaatagtggt gatagattta acgtggatct caattcatta
     2161 aagaggagaa aggtaccatg catcaccatc atcaccacag ccaggatcca atggtgagca
     2221 agggcgagga gaccacaatg ggcgtaatca agcccgacat gaagatcaag ctgaagatgg
     2281 agggcaacgt gaatggccac gccttcgtga tcgagggcga gggcgagggc aagccctacg
     2341 acggcaccaa caccatcaac ctggaggtga aggagggagc ccccctgccc ttctcctacg
     2401 acattctgac caccgcgttc agttacggca acagggcctt caccaagtac cccgacgaca
     2461 tccccaacta cttcaagcag tccttccccg agggctactc ttgggagcgc accatgacct
     2521 tcgaggacaa gggcatcgtg aaggtgaagt ccgacatctc catggaggag gactccttca
     2581 tctacgagat acacctcaag ggcgagaact tcccccccaa cggccccgtg atgcagaagg
     2641 agaccaccgg ctgggacgcc tccaccgaga ggatgtacgt gcgcgacggc gtgctgaagg
     2701 gcgacgtcaa gatgaagctg ctgctggagg gcggcggcca ccaccgcgtt gacttcaaga
     2761 ccatctacag ggccaagaag gcggtgaagc tgcccgacta tcactttgtg gaccaccgca
     2821 tcgagatcct gaaccacgac aaggactaca acaaggtgac cgtttacgag atcgccgtgg
     2881 cccgcaactc caccgacggc atggacgagc tgtacaaggg cgctgctaac gacgaaaact
     2941 acgctgacgc ttcttgaacg cactgaccac catgaaggtg acgctcttaa aaattaagcc
     3001 ctgaagaagg gcagcattca aagcagaagg ctttggggtg tgtgatacga aacgaagcat
     3061 tggttaaaaa taagcttgcg gccgcataat gcttaagtcg aacagaaagt aatcgtatga
     3121 gctcgacacc atcgaatggc gcaaaccttt cgcggtatgg catgatagcg cccggaagag
     3181 agtcaattca gggtggtgaa tgtataagaa ggagatatac atatgagcac aaaaaagaaa
     3241 ccattaacac aagagcagct tgaggacgca cgtcgcctta aagcaattta tgaaaaaaag
     3301 aaaaatgaac ttggcttatc ccaggaatct gtcgcagaca agatggggat ggggcagtca
     3361 ggcgttggtg ctttatttaa tggcatcaat gcattaaatg cttataacgc cgcattgctt
     3421 gcaaaaattc tcaaagttag cgttgaagaa tttagccctt caatcgccag agaaatctac
     3481 gagatgtatg aagcggttag tatgcagccg tcacttagaa gtgagtatga gtaccctgtt
     3541 ttttctcatg ttcaggcagg gatgttctca cctgagctta gaacctttac caaaggtgat
     3601 gcggagagat gggtaagcac aaccaaaaaa gccagtgatt ctgcattctg gcttgaggtt
     3661 gaaggtaatt ccatgaccgc accaacaggc tccaagccaa gctttcctga cggaatgtta
     3721 attctcgttg accctgagca ggctgttgag ccaggtgatt tctgcatagc cagacttggg
     3781 ggtgatgagt ttaccttcaa gaaactgatc agggatagcg gtcaggtgtt tttacaacca
     3841 ctaaacccac agtacccaat gatcccatgc aatgagagtt gttccgttgt ggggaaagtt
     3901 atcgctagtc agtggcctga agagacgttt ggctgactcg agtctggtaa agaaaccgct
     3961 gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta attaacctag
     4021 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     4081 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     4141 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4201 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4261 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4321 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     4381 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4441 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     4501 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4561 acaaaatatt aacgtttaca atttctggcg gcacgatggc atgagattat caaaaaggat
     4621 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4681 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4741 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4801 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4861 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4921 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4981 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     5041 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     5101 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     5161 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     5221 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     5281 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     5341 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5401 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5461 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5521 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatcatg
     5581 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5641 aaaataaaca aataggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5701 tcagaccccg tagaaaagat caaaggatct tc
//
