LOCUS       sequence-429314-        5105 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Ptrc99A-PAmCherry-L6-PopTag for bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5105)
  AUTHORS   Hoang Y, Azaldegui CA, Ghalmi M, Biteen JS, Vecchiarelli AG
  TITLE     An experimental framework to assess biomolecular condensates in 
            bacteria.
  JOURNAL   bioRxiv [Preprint]. 2023 Mar 23:2023.03.22.533878. doi: 
            10.1101/2023.03.22.533878.
  PUBMED    36993636
REFERENCE   2  (bases 1 to 5105)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2023 Mar 23:2023.03.22.533878. doi: 
            10.1101/2023.03.22.533878."
COMMENT     Sequence Label: pYH76
FEATURES             Location/Qualifiers
     source          1..5105
                     /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(1289..1306)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1459..1545
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1637..1664
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1683..1774
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1775..1843,1844..2635)
                     /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(1993..2012)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2806..3394
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3295..3314
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3548..3565
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3580..3720
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3646..3665)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3806..3828
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3906..3983
                     /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             3984..5066
                     /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(4003..4022)
                     /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 gcagcatggt cgcggaacaa cttgttggtg
     1021 tcagtgcggc ttccgctgcc gcagcgagcg cgttcggtag cttgagttct gctttattga
     1081 tgcctaagga tggtcgcacg ttggaggacg tggtacgtga gctgttgcgc ccgctgttaa
     1141 aggagtggct tgaccagaat ctgccccgta ttgtggagac taaagtggag gaagaggtac
     1201 aacgtatctc gcgtggtcgc ggagcttaat gagtcgacct gcaggcatgc aagcttggct
     1261 gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg
     1321 gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc
     1381 cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag
     1441 tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     1501 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat
     1561 ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc
     1621 aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact
     1681 cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1741 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     1801 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1861 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1921 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     1981 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact
     2041 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2101 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2161 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2221 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2281 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     2341 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2401 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2461 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2521 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2581 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2641 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     2701 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     2761 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2821 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2881 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2941 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3001 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3061 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3121 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3181 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3241 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3301 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3361 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3421 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3481 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3541 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3601 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga
     3661 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3721 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3781 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3841 tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcattt
     3901 acgttgacac catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     3961 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     4021 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     4081 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     4141 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     4201 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     4261 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     4321 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     4381 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     4441 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     4501 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     4561 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     4621 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     4681 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     4741 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
     4801 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     4861 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     4921 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     4981 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     5041 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gcgcgaattg
     5101 atctg
//
