LOCUS       sequence-244339-        8665 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses wild type R bodies with mNeonGreen-tagged second copy of 
            RebB under a weak RBS downstream as well as soluble mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8665)
  AUTHORS   Polka JK, Silver PA
  TITLE     A Tunable Protein Piston That Breaks Membranes to Release 
            Encapsulated Cargo.
  JOURNAL   ACS Synth Biol. 2016 Feb 3.
  PUBMED    26814170
REFERENCE   2  (bases 1 to 8665)
  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: "ACS Synth 
            Biol. 2016 Feb 3."
COMMENT     Sequence Label: Reb_206 aka PAS629
FEATURES             Location/Qualifiers
     source          1..8665
                     /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"
     CDS             complement(2016..3098)
                     /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
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVVVVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3485..3504
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3485..3503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3504..3528
                     /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)."
     CDS             7011..7031
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     primer_bind     complement(7084..7102)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7098..7145
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      7182..7637
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7269..7288)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7479..7500
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7729..8544)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(7729..8544)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     8452..8471
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8536..8555
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
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 taccgccttt 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 catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccaccac caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     3481 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagagaat
     3541 cagtacacac tttaaattaa ttcaaaacaa ggagctcaaa atggcgacta ctacttctac
     3601 tgctgatgta ggcaacacaa ctgacatcgt aaatagccaa attactgatt cagtaacaca
     3661 aactaacaca atggttcttg gttctactcc agcacaaatc atgacaaatt taatgcaaat
     3721 gagcgcacaa gctaatgctt tagcaatgca aaatgctgtt gttggtcaaa aacaaacaaa
     3781 tatgctatca gatgcagcaa ccactcaagg cttatctatt atgtatacgc tttcagctgc
     3841 ttcagatggt caagcagtca gtactgttga caattcaact gacatggctg gactgattga
     3901 tgcgcttggc gttgataaag cataagtatt aaaaatagat tatttataaa acaccgtaag
     3961 ataaaatctt acgatttaaa acccagacaa agtaaggaat aaaaaatgag taatgtaaat
     4021 tcacaaatta cagattcagt aacacaaaca aacactaaaa tattaggtga aatgcctgcg
     4081 tttaccactg gaagcttaat gcaaatggca acacaagctg ctggcttatc tatacaaaac
     4141 tctgtaacca atcagcaaca atctaatatg cttcatcagg catcaaccac tcaaggtatg
     4201 agtattctat attctgttga cacggcagct aatgctcaag ccattggttc agtgaaccgt
     4261 tctaatgaca caagccgttt aactgatgcc cttgctgtta ttaaagccgc taaaaatggt
     4321 taatgcttat ttcgatatgc taaaataatg ccaatcagat aaattggcta aattaaagta
     4381 tgataagcga aataaaatca tttaaagtag ataaaaatca tcagagacgg ttaattgttc
     4441 tcgtggggga agaaagctgg ggttaggaag tatacgttga tttgtattat tgagtttctc
     4501 ctcaacgcat aacgtattac ttcctgaccc aacgctatcg ttttccttat tttatttttt
     4561 tattttgctt tttgatatca atccatttta actagagctt caaactatgg cagataatgc
     4621 aataacaccc aatctttatg tgaatccaca agtgaccgat tctgtcactt caaatatgat
     4681 gaatgttata ggtatggcac cagctcaagc aatgggtgtg ctgtatcaag gcgttgcttc
     4741 aaccgctaat ttagctattc aaaatgctca atcaagctct caacagttaa atcaaatcgg
     4801 tcaagccgtc acttctgttg cctgtgaacg tattatgaaa atgatgggat aatcttgtaa
     4861 atggcggata tagaacattt ctcagaagat atcaatcaac ggatgatgga tcctaaaaat
     4921 aatgtcgcct ctcaaggtgc ttatgatatt gtttcaatgg ctgccgctag cgctgtgcaa
     4981 aatgaactgg ctatgcattc tggctttgaa acaacatcaa atacggtgct agcacttgct
     5041 atgcaaaaaa tggctgaagc tgccgcaatc gacaacgaac ctcaaattaa aatatgggaa
     5101 ggaatcatga aatccattag cagtaatcga gatgctgaag tggctaatct cagtaaattt
     5161 aatgagatga cacaaaactt agtgaaaaac tttccacgag gctaaaggag gtaaaaaatg
     5221 gttagtaaag gagaagaaaa taacatggca atcattaagg agttcatgag attcaaagtt
     5281 cacatggaag gttctgtaaa tggacatgaa tttgaaatag aaggtgaagg agaaggaagg
     5341 ccttatgaag gaacccaaac cgcgaagcta aaagttacta agggtggccc attaccattt
     5401 gcatgggata tccttagccc tcaattcatg tatgggtcaa aggcttatgt caagcacccc
     5461 gccgacattc cagactatct aaagttatct tttcccgaag ggtttaagtg ggagcgtgtg
     5521 atgaacttcg aagacggtgg cgtggtaaca gtgactcagg attcgtccct gcaagatggt
     5581 gaatttatct acaaagtcaa attaagagga actaactttc catctgacgg cccggttatg
     5641 caaaaaaaga caatgggctg ggaggcctcc tcagaacgaa tgtaccctga agatggtgcc
     5701 ttgaagggtg agattaaaca aagattgaaa ttgaaagatg gtggacatta tgacgctgag
     5761 gttaaaacga catacaaagc taagaaacct gtccagctcc caggtgctta caatgtaaat
     5821 ataaaacttg atattacatc acataatgaa gattatacga tagttgaaca atacgaaagg
     5881 gctgagggga gacatagtac tggtggcatg gatgaactat acaaaggttc tggtacagcg
     5941 taactcgagt cacacaggac tactagatgg ttagtaaggg tgaggaagac aatatggcaa
     6001 gcctgccggc aacgcacgaa ctgcatatct ttggttcaat caacggcgtg gactttgaca
     6061 tggtcggcca ggggactggt aacccgaacg atggatacga ggaactgaac ttaaaatcga
     6121 caaaaggaga tctgcaattc tcgccatgga ttctcgtacc gcatattggg tacggctttc
     6181 accagtattt gccgtatcca gatggcatga gtccgtttca agccgctatg gtcgatggta
     6241 gcggctatca ggtgcatcgc actatgcagt ttgaagacgg cgccagtttg acagtcaatt
     6301 accgttacac ttatgaaggc tctcatatta aaggtgaggc gcaagttaag ggtaccgggt
     6361 ttcccgccga cggtcccgta atgactaata gcttaaccgc ggctgattgg tgccgtagca
     6421 aaaaaaccta cccgaacgac aaaaccatca tttccacgtt taaatggagc tatactacag
     6481 gtaacgggaa gcgctatcgc tcaacggcgc gcactacata cacgttcgcg aaaccgatgg
     6541 ccgcgaatta cctcaaaaac cagccgatgt atgtatttcg taaaaccgag ctgaaacata
     6601 gcaagacaga actgaacttt aaagagtggc agaaagcatt cacagacgtc atgggaatgg
     6661 atgaacttta caagtcaggc caaggatccg gcatgagtaa tgtaaattca caaattacag
     6721 attcagtaac acaaacaaac actaaaatat taggtgaaat gcctgcgttt accactggaa
     6781 gcttaatgca aatggcaaca caagctgctg gcttatctat acaaaactct gtaaccaatc
     6841 agcaacaatc taatatgctt catcaggcat caaccactca aggtatgagt attctatatt
     6901 ctgttgacac ggcagctaat gctcaagcca ttggttcagt gaaccgttct aatgacacaa
     6961 gccgtttaac tgatgccctt gctgttatta aagccgctaa aaatggttaa caccaccacc
     7021 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     7081 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     7141 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     7201 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     7261 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     7321 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     7381 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     7441 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     7501 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     7561 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     7621 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     7681 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     7741 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     7801 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     7861 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     7921 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     7981 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     8041 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     8101 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     8161 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     8221 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     8281 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     8341 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     8401 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     8461 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     8521 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     8581 ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     8641 ccgtagaaaa gatcaaagga tcttc
//
