LOCUS       sequence-429300-        5327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTrc99A-mNeonGreen-mcdB for bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5327)
  AUTHORS   Hoang Y, Azaldegui CA, Dow RE, Ghalmi M, Biteen JS, Vecchiarelli AG
  TITLE     An experimental framework to assess biomolecular condensates in 
            bacteria.
  JOURNAL   Nat Commun. 2024 Apr 15;15(1):3222. doi: 10.1038/s41467-024-47330-4.
  PUBMED    38622124
REFERENCE   2  (bases 1 to 5327)
  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: "Nat 
            Commun."; date: "2024-04-15"; volume: "15(1):3222. doi"; pages: " 
            10.1038/s41467-024-47330-4"
COMMENT     Sequence Label: pYH74
FEATURES             Location/Qualifiers
     source          1..5327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(193..198,199..215,216..222)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    230..246
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     235..257
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     complement(1511..1528)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1681..1767
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1859..1886
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1905..1996
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1997..2065,2066..2857)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2215..2234)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3028..3616
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3517..3536
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3770..3787
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3802..3942
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3868..3887)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4028..4050
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4128..4205
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4206..5288
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4225..4244)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
       61 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
      121 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
      181 tgaaatgagc tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga
      241 taacaatttc acacaggaaa cagaccatgg cagtgtcgaa aggagaagaa gacaacatgg
      301 cgtcacttcc ggccacacat gaacttcaca ttttcggctc tattaacgga gtggatttcg
      361 acatggtcgg acaaggaaca gggaacccta acgacggata cgaggagtta aaccttaaaa
      421 gtacgaaggg agatttgcaa ttttcgcctt ggattttagt gcctcatatc ggttatggat
      481 tccaccagta cttgccgtat cctgatggca tgagtccgtt ccaggccgca atggttgatg
      541 gatcaggtta ccaagtacat cgcacgatgc agtttgaaga tggtgctagc ttaacagtga
      601 attatcgcta tacgtatgaa ggatctcata tcaaggggga ggctcaggtg aaagggacag
      661 gattcccggc agacggccct gtaatgacga actcactgac ggcagcggat tggtgtcgta
      721 gtaagaagac ctacccaaac gataaaacaa tcatctcaac atttaagtgg tcttatacca
      781 ctggtaatgg taagcgctac cgcagcacag cccgtacaac ctatactttt gctaagccga
      841 tggcggcaaa ttacttgaaa aaccagccca tgtatgtgtt tcgcaaaacc gaattgaagc
      901 attctaaaac ggaactgaat tttaaggagt ggcagaaggc gtttacggac gtgatgggga
      961 tggacgaatt atacaaggga tccggcagcg gcagcatgac tgacgcattc gaccgtctta
     1021 aaaaacgttc gcgtactccc attgcccgtg aagggtctct tacaacaggg cctgagctga
     1081 gcgaccgccc tttgcaactg ctgccacgtg agttcgaaac attttgcgac cgctacgcgg
     1141 tgcacgccgg cgacgtgatc gaagctgcgc ttgatttggt tttgcttgat ccagacttgc
     1201 aacaacgtct tcttcaacgt cttcgtcaag ggaatgggtc tgatcgcgtg tggttgggta
     1261 cggcttgccc tcgtagttgg caacagcagc tgcagcaaca agcccaggac caaggtctta
     1321 gtgaggccga tttactgcag gaggccattg cacaacgtct ggatcttgtc ctgggccaga
     1381 caacattgcg cgaggaggtg acactgcttc gccaagagct tgaccagtta aaacgcaagc
     1441 tgcacggttg gtgagtcgac ctgcaggcat gcaagcttgg ctgttttggc ggatgagaga
     1501 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt
     1561 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac
     1621 gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat
     1681 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     1741 gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa
     1801 cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag
     1861 aaggccatcc tgacggatgg cctttttgcg tttctacaaa ctcttttgtt tatttttcta
     1921 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1981 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     2041 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     2101 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     2161 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     2221 tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     2281 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     2341 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     2401 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     2461 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     2521 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     2581 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     2641 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     2701 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     2761 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     2821 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     2881 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     2941 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     3001 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     3061 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3121 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     3181 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3241 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3301 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3361 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3421 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3481 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3541 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3601 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3661 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3721 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3781 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3841 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3901 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3961 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4021 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     4081 cagcagatca attcgcgcgc gaaggcgaag cggcatgcat ttacgttgac accatcgaat
     4141 ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     4201 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     4261 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     4321 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     4381 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     4441 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     4501 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     4561 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     4621 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     4681 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     4741 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     4801 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     4861 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     4921 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     4981 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     5041 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     5101 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     5161 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     5221 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     5281 ggcagtgagc gcaacgcaat taatgtaagt tagcgcgaat tgatctg
//
