LOCUS       sequence-323485-        4416 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lactate-inducible expression of green fluorescent protein encoding 
            zero amber codons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4416)
  AUTHORS   Ho JML, Miller CA, Parks SE, Mattia JR, Bennett MR
  TITLE     A suppressor tRNA-mediated feedforward loop eliminates leaky gene 
            expression in bacteria.
  JOURNAL   Nucleic Acids Res. 2021 Mar 18;49(5):e25. doi: 10.1093/nar/gkaa1179.
  PUBMED    33290521
REFERENCE   2  (bases 1 to 4416)
  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: "Nucleic 
            Acids Res."; date: "2021-03-18"; volume: "49(5):e25. doi"; pages: " 
            10.1093/nar/gkaa1179"
COMMENT     Sequence Label: pJH625_pJH_LldR_PlldR-sfGFP.0TAG
FEATURES             Location/Qualifiers
     source          1..4416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(59..127,128..919)
                     /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     complement(277..296)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1090..1678
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1579..1598
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1768..1785)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1938..2024
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2116..2143
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             2479..3192
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     RBS             3485..3493
                     /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      4331..4378
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
       61 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
      121 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
      181 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
      241 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      301 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      361 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      421 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
      481 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
      541 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
      601 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
      661 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
      721 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
      781 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
      841 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
      901 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
      961 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     1021 caaaatccct taacgtgggt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1081 aggatcatct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1141 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1201 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     1261 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1321 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1381 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1441 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1501 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1561 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     1621 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     1681 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcatggctg
     1741 ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg
     1801 tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc
     1861 gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt
     1921 agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     1981 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
     2041 tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
     2101 ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc
     2161 tgctagcaag taaggccgac caacaaccta agggcaattc tctgatgagg attgcccttt
     2221 tctttaccag acatctcccc ccacaagaat tggccctacc aattcttcgc ttatctgacc
     2281 tctggttcac aatttcccaa ttaaaactca catcaatgtt gccaatacat aacatttagt
     2341 taaccattca ttgtcattat ccctacacaa cacaattggc agtgccactt ttacacaacg
     2401 tgtgacaagg agatgagcaa cagactcatt acacgatgtg cgtggactcc catttaactt
     2461 taagaaggag atatacatat gcgtaaaggt gaagaactgt tcaccggtgt tgttccgatc
     2521 ctggttgaac tggacggtga cgttaacggt cacaaattct ctgttcgtgg tgaaggtgaa
     2581 ggtgacgcta ccaacggtaa actgaccctg aaattcatct gcaccaccgg taaactgccg
     2641 gttccgtggc cgaccctggt taccaccctg acctacggtg ttcagtgctt cgctcgttac
     2701 ccggaccaca tgaaacagca cgacttcttc aaatctgcta tgccggaagg ttacgttcag
     2761 gaacgtacca tctctttcaa agacgacggt acctacaaaa cccgtgctga agttaaattc
     2821 gaaggtgaca ccctggttaa ccgtatcgaa ctgaaaggta tcgacttcaa agaagacggt
     2881 aacatcctgg gtcacaaact ggaatacaac ttcaactctc acaacgttta catcaccgct
     2941 gacaaacaga aaaacggtat caaagctaac ttcaaaatcc gtcacaacgt tgaagacggt
     3001 tctgttcagc tggctgacca ctaccagcag aacaccccga tcggtgacgg tccggttctg
     3061 ctgccggaca accactacct gtctacccag tctgttctgt ctaaagaccc gaacgaaaaa
     3121 cgtgaccaca tggttctgct ggaattcgtt accgctgctg gtatcaccca cggtatggac
     3181 gaactgtaca aagatgcatg ccagttctaa cataacccta atgagtgagc taacttacat
     3241 taattgcgtt gcgccttaat taacggcact cctcagcaaa tataatgacc ctcttgataa
     3301 cccaagaggg cattttttaa tgcccatggc gtttaccaca gctaacacca cgtcgtccct
     3361 atctgctgcc ctaggtctat gagtggttgc tggataactt tacgggcatg cataaggctc
     3421 gtaggctata ttcagggaga ccacaacggt ttccctctac aaataatttt gtttaacttt
     3481 gaaataagga ggtaatacaa atgattgttt tacccagacg cctgtcagac gaggttgccg
     3541 atcgtgtgcg ggcgctgatt gatgaaaaaa acctggaagc gggcatgaag ttgcccgctg
     3601 agcgccaact ggcgatgcaa ctcggcgtat cacgtaattc actgcgcgag gcgctggcaa
     3661 aactggtgag tgaaggcgtg ctgctcagtc gacgcggcgg cgggacgttt attcgctggc
     3721 gtcatgacac atggtcggag caaaacatcg tccagccgct aaaaacactg atggccgatg
     3781 atccggatta cagtttcgat attctggaag cccgctacgc cattgaagcc agcaccgcat
     3841 ggcatgcggc aatgcgcgcc acacctggcg acaaagaaaa gattcagctt tgctttgaag
     3901 caacgctaag tgaagacccg gatatcgcct cacaagcgga cgttcgtttt catctggcga
     3961 ttgccgaagc ctcacataac atcgtgctgc tgcaaaccat gcgcggtttc ttcgatgtcc
     4021 tgcaatcctc agtgaagcat agccgtcagc ggatgtatct ggtgccaccg gttttttcac
     4081 aactgaccga acaacatcag gctgtcattg acgccatttt tgccggtgat gctgacgggg
     4141 cgcgtaaagc aatgatggcg caccttagtt ttgttcacac caccatgaaa cgattcgatg
     4201 aagatcaggc tcgccacgca cggattaccc gcctgcccgg tgagcataat gagcattcga
     4261 gggagaaaaa cgcataataa gcgactaaaa aattgaatgt aggaaaccaa catgccagtt
     4321 cgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     4381 gaaacctcag gcatttgaga agcacacggt cacact
//
