LOCUS       sequence-426588-        5018 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Always-off, unactivated expression of mWasabi reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5018)
  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 5018)
  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: pBaseline-PRM-wasabi
FEATURES             Location/Qualifiers
     source          1..5018
                     /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             3235..3279
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3317..3335)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3331..3378
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3415..3870
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3502..3521)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3712..3733
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3985..4776,4777..4845))
                     /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     4608..4627
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4846..4938)
                     /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 tacagccttt 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 atctcgagtc tggtaaagaa
     3241 accgctgctg cgaaatttga acgccagcac atggactcgt ctactagcgc agcttaatta
     3301 acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     3361 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     3421 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3481 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3541 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3601 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3661 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3721 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3781 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3841 attttaacaa aatattaacg tttacaattt ctggcggcac gatggcatga gattatcaaa
     3901 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3961 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4021 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4081 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4141 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4201 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4261 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4321 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4381 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4441 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4501 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4561 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4621 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4681 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4741 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4801 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4861 atcatgattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4921 tttagaaaaa taaacaaata ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4981 tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
