LOCUS       sequence-429319-        5351 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTrc99A-PAmCherry-cI78EP8 for bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5351)
  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 5351)
  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: pCA3
FEATURES             Location/Qualifiers
     source          1..5351
                     /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"
     CDS             267..977
                     /codon_start=1
                     /product="photoactivatable monomeric derivative of DsRed 
                     fluorescent protein"
                     /label=PAmCherry
                     /note="mammalian codon-optimized"
                     /translation="MAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHVFEIEGEGEGRPYE
                     GTQTAKLKVTKGGPLPFTWDILSPQFMYGSNAYVKHPADIPDYFKLSFPEGFKWERVMK
                     FEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEALSERMYPEDGAL
                     KGEVKPRVKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNRKLDITSHNEDYTIVEQYER
                     AEGRHSTGGMDELYK"
     primer_bind     complement(415..433)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     683..702
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1535..1552)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1705..1791
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1883..1910
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1929..2020
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2021..2089,2090..2881)
                     /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(2239..2258)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3052..3640
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3541..3560
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3794..3811
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3826..3966
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3892..3911)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4052..4074
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4152..4229
                     /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             4230..5312
                     /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(4249..4268)
                     /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 cagtgagcaa gggcgaggag gataacatgg
      301 ccatcattaa ggagttcatg cgcttcaagg tgcacatgga ggggtccgtg aacggccacg
      361 tgttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
      421 tgaaggtgac caagggtggc cccctgccct tcacctggga catcctgtcc cctcaattca
      481 tgtacggctc caatgcctac gtgaagcacc ccgccgacat ccccgactac tttaagctgt
      541 ccttccccga gggcttcaag tgggagcgcg tgatgaaatt cgaggacggc ggcgtggtga
      601 ccgtgaccca ggactcctcc ctgcaggacg gtgagttcat ctacaaggtg aagctgcgcg
      661 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggccc
      721 tctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgaggtcaag ccgagagtga
      781 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
      841 ccgtgcagct gcccggcgcc tacaacgtca accgcaagtt ggacatcacc tcacacaacg
      901 aggactacac catcgtggaa cagtacgaac gtgccgaggg ccgccactcc accggcggca
      961 tggacgagct gtacaaggga tccggcagcg gcagcagccc ttcaatcgcc agagaaatct
     1021 acgagatgta tgtagcggtt agtatgcagc cgtcacttag aagtgagtat gagtaccctg
     1081 ttttttctca tgttcaggca gggatgttct cacctgagct tagaacctta accaaaggtg
     1141 atgcggagag atgggtaagc acaaccaaaa aagctagtga ttctgcattc tggcttgagg
     1201 ttgaaggtaa ttccatgacc gcaccaacag gctccaagcc aagctttcct gacggaatgt
     1261 taattctcgt tgaccctgag caggctgttg agccaggtga tttctgcata gccagacttg
     1321 ggggtgatga gtttaccctc aagaaactga tcagggatag cggtcaggtg tttttacaac
     1381 cactaaaccc acagtaccca atgatcccat gcaatgagag ttgttccgtt gtggggaaag
     1441 ttatcgctag tcagtggcct gaagagacgt ttggctgagt cgacctgcag gcatgcaagc
     1501 ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca
     1561 gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc
     1621 ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg
     1681 cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     1741 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga
     1801 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa
     1861 actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta
     1921 caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1981 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     2041 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     2101 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     2161 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     2221 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     2281 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     2341 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     2401 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     2461 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     2521 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     2581 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2641 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2701 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     2761 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     2821 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     2881 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     2941 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     3001 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3061 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3121 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3181 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3241 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3301 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3361 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3421 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3481 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3541 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3601 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3661 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3721 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3781 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3841 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     3901 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3961 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4021 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4081 ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc gcgcgaaggc gaagcggcat
     4141 gcatttacgt tgacaccatc gaatggtgca aaacctttcg cggtatggca tgatagcgcc
     4201 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     4261 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     4321 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     4381 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     4441 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     4501 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     4561 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     4621 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     4681 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     4741 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     4801 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     4861 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     4921 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     4981 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     5041 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     5101 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     5161 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     5221 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     5281 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagcgc
     5341 gaattgatct g
//
