LOCUS       sequence-148760-        9342 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Output plasmid: contains 5-state register that regulates expression 
            of GFP. Doesn't behave as expected..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9342)
  AUTHORS   Roquet N, Soleimany AP, Ferris AC, Aaronson S, Lu TK
  TITLE     Synthetic recombinase-based state machines in living cells.
  JOURNAL   Science. 2016 Jul 22;353(6297):aad8559. doi: 
            10.1126/science.aad8559. Epub 2016 Jul 21.
  PUBMED    27463678
REFERENCE   2  (bases 1 to 9342)
  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: "Science."; 
            date: "2016-07-22"; volume: "353(6297):aad8559. doi"; pages: " 
            10.1126/science.aad8559. Epub 2016 Jul 21"
COMMENT     Sequence Label: pNR166
FEATURES             Location/Qualifiers
     source          1..9342
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        505..554
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             597..605
                     /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)"
     primer_bind     complement(641..669)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1264..1284
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    1426..1474
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     terminator      complement(1699..1770)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    1914..1971
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attP site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     misc_feature    2686..3159
                     /gene="sopC"
                     /label=sopC
                     /note="centromere-like partitioning region of the bacterial
                     F plasmid"
     CDS             complement(3232..4203)
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     CDS             complement(4203..5378)
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     misc_feature    5704..5954
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(5957..6712)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      6803..7022
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     rep_origin      7090..7621
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      7098..7712
                     /label=oriV
                     /note="origin of replication for the bacterial F plasmid"
     promoter        8570..8672
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             8673..9332
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(8739..8758)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 taagtgccca ttcagccagt ataaccagat aaaaaaaatc cttagctttc gctaaggatg
       61 atttctgtca gccaggaagg tataacagga aaaattaaag gggagataaa atcccccctt
      121 tttggttaac tcgaggattc gaaaaggtga aatctccttt cacggcccat tcctcatgga
      181 tgggccgttt atttccccga gccagacgtc caccatacct tcgatattcg cgcccactct
      241 cccattagtc ggagtggatg tgttgcgatt gcccgctaga tcaatgtgca cgtcgagtgc
      301 ggtattcctc tgggcatgcc aacacaatta acatctcaat caaggtaaat gctttttgct
      361 ttttttgccg tggtgcggat ctagctaccc gcacttgagt ccacggctga tgccaacaca
      421 attaacatca gaatcaaggt aaatgctttt tgcttttttt gcgggctcac tagggaatgg
      481 cggtgaggac gagggatggc acagctgtca ccggatgtgc tttccggtct gatgagtccg
      541 tgaggacgaa acagcctcta caaataattt tgtttaaggt cgaagccgac ctatattaag
      601 gaggtcacta atgagtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga
      661 attagatggt gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc
      721 aacatacgga aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg
      781 gccaacactt gtcactactt tcggttatgg tgttcaatgc tttgcgagat acccagatca
      841 tatgaaacag catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac
      901 tatatttttc aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga
      961 tacccttgtt aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct
     1021 tggacacaaa ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca
     1081 aaagaatgga atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca
     1141 actagcagac cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga
     1201 caaccattac ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca
     1261 catggtcctt cttgagtttg taacagctgc tgggattaca catggcatgg atgatctcta
     1321 caaataagga aacacagaaa aaagcccgca cctgacagtg cgggcttttt ttttcgacca
     1381 aagggtgctt tatcaagggg aaggtcgtcc gtgacattct gtgcgcggcc ggcttgtcga
     1441 cgacggcggt ctccgtcgtc aggatcatcc gggccagcgg ctgtctatgc cagaagccac
     1501 cgggtagctc aatgcgcgca ataaaaaagc ccccggaagg tgatcttccg ggggctttct
     1561 catgcgttgg tgttcactcc cattcgggtt gagaagagaa aagaaaaccg ccgatcctgt
     1621 ccaccgcatt actgcaaggt agtggacaag accggcggtc ttaagttttt tggctgaact
     1681 taagctgcga gtgggcccga gagcgttcac cgacaaacaa cagataaaac gaaaggccca
     1741 gtctttcgac tgagcctttc gttttatttg atgcctgggg agtgtgagtg ggaggtctgt
     1801 tcggtcaggt cggagtccta gaaaggagtt ttttagttac cttaattcta ataaacgaaa
     1861 taaaaactcg ccgttcgcgc gacatgacat gccgacacaa tctccctgct caggtcgtgg
     1921 tttgtctggt caaccaccgc ggtctcagtg gtgtacggta caaaccccga cgcaatagcg
     1981 ccaaccatgc actcggcgtt aatccttgtt cactatatgc aaccattatc accgccagag
     2041 gtaaaatagt caaacgcacg gtgttagcca tagccagcct gacagtagcc aaaggagttt
     2101 tttagttacc ttaattgaaa taaacgaaat aaaaactcgc gcagctgaga gcgacacagt
     2161 cgccgccgat tgattcagaa agttaaacaa aattatttgt agagggaaac cgttgtggtc
     2221 tccctgaata tattatacga gccttatgca tgcccgtaaa gttatccagc aaccactcat
     2281 agacctaggg cagcagatag ggacgacgtg gtgttagctg tgtgcctcac tcctagggtg
     2341 taataggcac taaaagagtg atatggcacg cgcatgtgga acgccgttag agggttgctg
     2401 agccatcgtg gtccttagac tcgggaaaaa caaaaacccg ccgaagcggg ttaagtgcgg
     2461 ggcggctagc ccgccaccta agttatgagt caggaaaaaa ggcgacagag taatctgtcg
     2521 ccttttttct ttgcttgctt tgtaatcact cgatgaccaa aagtaaaaac ccgccgaagc
     2581 gggtttttac gtaaaacagg tgaaactgac gctagccggt cacatgtcaa tacttgccct
     2641 tgacaggcat tgatggaatc gtagtctcac gctgatagtc tgatcgacaa tacaagtggg
     2701 accgtggtcc cagaccgata atcagaccga caacacgagt gggatcgtgg tcccagacta
     2761 ataatcagac cgacgatacg agtgggaccg tggtcccaga ctaataatca gaccgacgat
     2821 acgagtggga ccgtggttcc agactaataa tcagaccgac gatacgagtg ggaccgtggt
     2881 cccagactaa taatcagacc gacgatacga gtgggaccat ggtcccagac taataatcag
     2941 accgacgata cgagtgggac cgtggtccca gtctgattat cagaccgacg atacgagtgg
     3001 gaccgtggtc ccagactaat aatcagaccg acgatacgag tgggaccgtg gtcccagact
     3061 aataatcaga ccgacgatac gagtgggacc gtggtcccag tctgattatc agaccgacga
     3121 tacaagtgga acagtgggcc cagagagaat attcaggcca gttatgcttt ctggcctgta
     3181 acaaaggaca ttaagtaaag acagataaac gtagactaaa acgtggtcgc atcagggtgc
     3241 tggcttttca agttccttaa gaatggcctc aattttctct atacactcag ttggaacacg
     3301 ggacctgtcc aggttaagca ccattttatc gcccttatac aatactgtcg ctccaggagc
     3361 aaactgatgt cgtgagctta aactagttct tgatgcagat gacgttttaa gcacagaagt
     3421 taaaagagtg ataacttctt cagcttcaaa tatcacccca gcttttttct gctcatgaag
     3481 gttagatgcc tgctgcttaa gtaattcctc tttatctgta aaggcttttt gaagtgcatc
     3541 acctgaccgg gcagatagtt caccggggtg agaaaaaaga gcaacaactg atttaggcaa
     3601 tttggcggtg ttgatacagc gggtaataat cttacgtgaa atattttccg catcagccag
     3661 cgcagaaata tttccagcaa attcattctg caatcggctt gcataacgct gaccacgttc
     3721 ataagcactt gttgggcgat aatcgttacc caatctggat aatgcagcca tctgctcatc
     3781 atccagctcg ccaaccagaa cacgataatc actttcggta agtgcagcag ctttacgacg
     3841 gcgactccca tcggcaattt ctatgacacc agatactctt cgaccgaacg ccggtgtctg
     3901 ttgaccagtc agtagaaaag aagggatgag atcatccagt gcgtcctcag taagcagctc
     3961 ctggtcacgt tcattacctg accatacccg agaggtcttc tcaacactat caccccggag
     4021 cacttcaaga gtaaacttca catcccgacc acatacaggc aaagtaatgg cattaccgcg
     4081 agccattact cctacgcgcg caattaacga atccaccatc ggggcagctg gtgtcgataa
     4141 cgaagtatct tcaaccggtt gagtattgag cgtatgtttt ggaataacag gcgcacgctt
     4201 cattatctaa tctcccagcg tggtttaatc agacgatcga aaatttcatt gcagacaggt
     4261 tcccaaatag aaagagcatt tctccaggca ccagttgaag agcgttgatc aatggcctgt
     4321 tcaaaaacag ttctcatccg gatctgacct ttaccaactt catccgtttc acgtacaaca
     4381 ttttttagaa ccatgcttcc ccaggcatcc cgaatttgct cctccatcca cggggactga
     4441 gagccattac tattgctgta tttggtaagc aaaatacgta catcaggctc gaacccttta
     4501 agatcaacgt tcttgagcag atcacgaagc atatcgaaaa actgcagtgc ggaggtgtag
     4561 tcaaacaact cagcaggcgt gggaacaatc agcacatcag cagcacatac gacattaatc
     4621 gtgccgatac ccaggttagg cgcgctgtca ataactatga catcatagtc atgagcaaca
     4681 gtttcaatgg ccagtcggag catcaggtgt ggatcggtgg gcagtttacc ttcatcaaat
     4741 ttgcccatta actcagtttc aatacggtgc agagccagac aggaaggaat aatgtcaagc
     4801 cccggccagc aagtgggctt tattgcataa gtgacatcgt ccttttcccc aagatagaaa
     4861 ggcaggagag tgtcttctgc atgaatatga agatctggta cccatccgtg atacattgag
     4921 gctgttccct gggggtcgtt accttccacg agcaaaacac gtagcccctt cagagccaga
     4981 tcctgagcaa gatgaacaga aactgaggtt ttgtaaacgc cacctttatg ggcagcaacc
     5041 ccgatcaccg gtggaaatac gtcttcagca cgtcgcaatc gcgtaccaaa cacatcacgc
     5101 atatgattaa tttgttcaat tgtataacca acacgttgct caacccgtcc tcgaatttcc
     5161 atatccgggt gcggtagtcg ccctgctttc tcggcatctc tgatagcctg agaagaaacc
     5221 ccaactaaat ccgctgcttc acctattctc cagcgccggg ttattttcct cgcttccggg
     5281 ctgtcatcat taaactgtgc aatggcgata gccttcgtca tttcatgacc agcgtttatg
     5341 cactggttaa gtgtttccat gagtttcatt ctgaacatcc tttaatcatt gctttgcgtt
     5401 tttttattaa atcttgcaat ttactgcaaa gcaacaacaa aatcgcaaag tcatcaaaaa
     5461 accgcaaagt tgtttaaaat aagagcaaca ctacaaaagg agataagaag agcacatacc
     5521 tcagtcactt attatcacta gcgctcgccg cagccgtgta accgagcata gcgagcgaac
     5581 tggcgaggaa gcaaagaaga actgttctgt cagatagctc ttacgctcag cgcaagaaga
     5641 aatatccacc gtgggaaaaa ctccaggtag aggtacacac gcggatagcc aattcagagt
     5701 aataaactgt gataatcaac cctcatcaat gatgacgaac taacccccga tatcaggtca
     5761 catgacgaag ggaaagagaa ggaaatcaac tgtgacaaac tgccctcaaa tttggcttcc
     5821 ttaaaaatta cagttcaaaa agtatgagaa aatccatgca ggctgaagga aacagcaaaa
     5881 ctgtgacaaa ttaccctcag taggtcagaa caaatgtgac gaaccaccct caaatctgtg
     5941 acagataacc ctcagactat cctgtcgtca tggaagtgat atcgcggaag gaaaatacga
     6001 tatgagtcgt ctggcggcct ttctttttct caatgtatga gaggcgcatt ggagttctgc
     6061 tgttgatctc attaacacag acctgcagga agcggcggcg gaagtcaggc atacgctggt
     6121 aactttgagg cagctggtaa cgctctatga tccagtcgat tttcagagag acgatgcctg
     6181 agccatccgg cttacgatac tgacacaggg attcgtataa acgcatggca tacggattgg
     6241 tgatttcttt tgtttcacta agccgaaact gcgtaaaccg gttctgtaac ccgataaaga
     6301 agggaatgag atatgggttg atatgtacac tgtaaagccc tctggatgga ctgtgcgcac
     6361 gtttgataaa ccaaggaaaa gattcatagc ctttttcatc gccggcatcc tcttcagggc
     6421 gataaaaaac cacttccttc cccgcgaaac tcttcaatgc ctgccgtata tccttactgg
     6481 cttccgcaga ggtcaatccg aatatttcag catatttagc aacatggatc tcgcagatac
     6541 cgtcatgttc ctgtagggtg ccatcagatt ttctgatctg gtcaacgaac agatacagca
     6601 tacgtttttg atcccgggag agactatatg ccgcctcagt gaggtcgttt gactggacga
     6661 ttcgcgggct atttttacgt ttcttgtgat tgataaccgc tgtttccgcc atgacagatc
     6721 catgtgaagt gtgacaagtt tttagattgt cacactaaat aaaaaagagt caataagcag
     6781 ggataacttt gtgaaaaaac agcttcttct gagggcaatt tgtcacaggg ttaagggcaa
     6841 tttgtcacag acaggactgt catttgaggg tgatttgtca cactgaaagg gcaatttgtc
     6901 acaacacctt ctctagaacc agcatggata aaggcctaca aggcgctcta aaaaagaaga
     6961 tctaaaaact ataaaaaaaa taattataaa aatatccccg tggataagtg gataacccca
     7021 agggaagttt tttcaggcat cgtgtgtaag cagaatatat aagtgctgtt ccctggtgct
     7081 tcctcgctca ctcgaccggg agggttcgag aagggggggc accccccttc ggcgtgcgcg
     7141 gtcacgcgca cagggcgcag ccctggttaa aaacaaggtt tataaatatt ggtttaaaag
     7201 caggttaaaa gacaggttag cggtggccga aaaacgggcg gaaacccttg caaatgctgg
     7261 attttctgcc tgtggacagc ccctcaaatg tcaataggtg cgcccctcat ctgtcagcac
     7321 tctgcccctc aagtgtcaag gatcgcgccc ctcatctgtc agtagtcgcg cccctcaagt
     7381 gtcaataccg cagggcactt atccccaggc ttgtccacat catctgtggg aaactcgcgt
     7441 aaaatcaggc gttttcgccg atttgcgagg ctggccagct ccacgtcgcc ggccgaaatc
     7501 gagcctgccc ctcatctgtc aacgccgcgc cgggtgagtc ggcccctcaa gtgtcaacgt
     7561 ccgcccctca tctgtcagtg agggccaagt tttccgcgag gtatccacaa cgccggcggc
     7621 cggccgcggt gtctcgcaca cggcttcgac ggcgtttctg gcgcgtttgc agggccatag
     7681 acggccgcca gcccagcggc gagggcaacc agccgagggc ttcgccctgt cgctcgactg
     7741 cggcgagcac tactggctgt aaaaggacag accacatcat ggttctgtgt tcattaggtt
     7801 gttctgtcca ttgctgacat aatccgctcc acttcaacgt aacaccgcac gaagatttct
     7861 attgttcctg aaggcatatt caaatcgttt tcgttaccgc ttgcaggcat catgacagaa
     7921 cactacttcc tataaacgct acacaggctc ctgagattaa taatgcggat ctctacgata
     7981 atgggagatt ttcccgactg tttcgttcgc ttctcagtgg ataacagcca gcttctctgt
     8041 ttaacagaca aaaacagcat atccactcag ttccacattt ccatataaag gccaaggcat
     8101 ttattctcag gataattgtt tcagcatcgc aaccgcatca gactccggca tcgcaaactg
     8161 cacccggtgc cgggcagcca catccagcgc aaaaaccttc gtgtagactt ccgttgaact
     8221 gatggactta tgtcccatca ggctttgcag aactttcagc ggtataccgg catacagcat
     8281 gtgcatcgca taggaatggc ggaacgtatg tggtgtgacc ggaacagaga acgtcacacc
     8341 gtcagcagca gcggcggcaa ccgcctcccc aatccaggtc ctgaccgttc tgtccgtcac
     8401 ttcccagatc cgcgctttct ctgtccttcc tgtgcgacgg ttacgccgct ccatgagctt
     8461 atcgcgaata aatacctgtg acggaagatc acttcgcaga ataaataaat cctggtgtcc
     8521 ctgttgatac cgggaagccc tgggccaact tttggcgaaa atgagacgtt gatcggcacg
     8581 taagaggttc caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt
     8641 tatcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca
     8701 ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc
     8761 aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga
     8821 aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc
     8881 atccggaatt tcgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc
     8941 cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc
     9001 acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa
     9061 acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct
     9121 gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg
     9181 ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc
     9241 aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac
     9301 agtactgcga tgagtggcag ggcggggcgt aagacgtcta ag
//
