LOCUS       sequence-313946-        9337 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Nanoluc placed on Output 2 of the 2in3out circuit (A001 and P002 
            design) with aTC and Bile Acid as inputs, AmpR, ErmR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9337)
  AUTHORS   Taketani M, Zhang J, Zhang S, Triassi AJ, Huang YJ, Griffith LG, 
            Voigt CA
  TITLE     Genetic circuit design automation for the gut resident species 
            Bacteroides thetaiotaomicron.
  JOURNAL   Nat Biotechnol. 2020 Aug;38(8):962-969. doi: 
            10.1038/s41587-020-0468-5. Epub 2020 Mar 30.
  PUBMED    32231334
REFERENCE   2  (bases 1 to 9337)
  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"; doi: 
            "10.1038/s41587-020-0468-5"; journalName: "Nat Biotechnol"; date: 
            "2020-08"; volume: "38"; issue: "8"; pages: "962-969"
COMMENT     Sequence Label: pMT468
FEATURES             Location/Qualifiers
     source          1..9337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      132..520
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    709..727
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    771..789
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        814..889
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1050..1068
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1112..1130
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        1155..1230
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        1441..1516
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        1723..1798
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        2017..2092
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        2307..2382
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        2570..2619
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     misc_RNA        2891..2940
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     misc_RNA        3212..3261
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(8198..8266,8267..9058)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(8416..8435)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     oriT            9189..9298
                     /note="incP origin of transfer"
ORIGIN
        1 accctttggc aaaatcctgt atatcgtgcg aaaaaggatg gatataccga aaaaatcgct
       61 ataatgaccc cgaagcaggg ttatgcagcg gaaaacggaa ttgatccggc cacgatgcgt
      121 ccggcgtaga ggatctgaag atcagcagtt caacctgttg atagtacgta ctaagctctc
      181 atgtttcacg tactaagctc tcatgtttaa cgtactaagc tctcatgttt aacgaactaa
      241 accctcatgg ctaacgtact aagctctcat ggctaacgta ctaagctctc atgtttcacg
      301 tactaagctc tcatgtttga acaataaaat taatataaat cagcaactta aatagcctct
      361 aaggttttaa gttttataag aaaaaaaaga atatataagg cttttaaagc ttttaaggtt
      421 taacggttgt ggacaacaag ccagggatgt aacgcactga gaagccctta gagcctctca
      481 aagcaatttt gagtgacaca ggaacactta acggctgaca tgggaattcc cctccaccgc
      541 ggtgtattaa tgcggctgca atttttttgg gcggggccgc ccaaaaaaat cctagcaccc
      601 tgcagggtct cccgctggag ttttgcaccc gctttccaag agaagaaagc cttgttaaat
      661 tgacttagtg taaaagcgca gtactgcttg accataagaa caaaaaaatc tctatcactg
      721 atagggataa agtttggaag ataaagctaa aagttcttat ctttgcagtc tccctatcag
      781 tgatagagac gcacaaagat aatagaactt aaggttttag agctagaaat agcaagttaa
      841 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttggat
      901 ccaaaaaaaa aaaacaccct aacgggtgtt tttttttttt tggtctgccg cggccgctac
      961 gttttgcacc cgctttccaa gagaagaaag ccttgttaaa ttgacttagt gtaaaagcgc
     1021 agtactgctt gaccataaga acaaaaaaat ctctatcact gatagggata aagtttggaa
     1081 gataaagcta aaagttctta tctttgcagt ctccctatca gtgatagaga cgcacaaaga
     1141 taattaagtt aagggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     1201 caacttgaaa aagtggcacc gagtcggtgc tttttttgga tccctcggta ccaaattcca
     1261 gaaaagagac gctgaaaagc gtcttttttc gttttggtcc gcggccgcag gttgctgtca
     1321 taggccggtt gtccggtagt ccggatgatt taaggaagat ttctcttctt ttttcacttt
     1381 ctccgaacaa cctccactta agttctatta tctttgcagt caaagatact tatgttagaa
     1441 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     1501 ggcaccgagt cggtgctttt tttggatcca acgcatgaga aagcccccgg aagatcacct
     1561 tccgggggct tttttattgc gcgcggccgc gctttgctgt cataggccgg ttgtccggta
     1621 gtccggatga tttaaggaag atttctcttc ttttttcact ttctccgaac aacctccact
     1681 taagttctat tatctttgca gtcaaagata attaagttaa gggttttaga gctagaaata
     1741 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     1801 tttttggatc cctcggtacc aaattccaga aaagagacgc tgaaaagcgt cttttttcgt
     1861 tttggtccgc ggccgccgct ttacaaagaa aattcgacaa actgttattt ttctatctat
     1921 ttaaatgtac acccgtttgt ttactttact ttagttatgt acctttgtcg gcaatgtaca
     1981 cccgaaagtt tactttcaaa gatacttatg ttagaagttt tagagctaga aatagcaagt
     2041 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg
     2101 gatccaacgc atgagaaagc ccccggaaga tcaccttccg ggggcttttt tattgcgcgc
     2161 ggccgctgtc ttacaaagaa aattcgacaa actgttattt ttctatctat ttaaatgtac
     2221 acccgtttgt ttactttact ttagttatgt acctttgtcg gcaatgtaca cccgaaagtt
     2281 tactttcaaa gataatagaa cttaaggttt tagagctaga aatagcaagt taaaataagg
     2341 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg gatccaaaaa
     2401 aaaaaaacac cctaacgggt gttttttttt tttttggtct gccgcggccg caatgggagt
     2461 gctgtcatag gccggttgtc cggtagtccg gatgatttaa ggaagatttc tcttcttttt
     2521 tcactttctc cgaacaacct ccattctaac ataagtatct ttgcagtagc tgtcaccgga
     2581 tgtgctttcc ggtctgatga gtccgtgagg acgaaacagc ctctacaaat aattttgttt
     2641 aacgcatttt aaaataaaat aaattattta tttaattaaa cgaatatgta atgaactggg
     2701 atccctcggt accaaatttt cgaaaaaaga cgctgaaaag cgtctttttt cgttttggtc
     2761 cgcggccgcc ggccgctacg tgctgtcata ggccggttgt ccggtagtcc ggatgattta
     2821 aggaagattt ctcttctttt ttcactttct ccgaacaacc tccacttaag ttctattatc
     2881 tttgcagtag ctgtcaccgg atgtgctttc cggtctgatg agtccgtgag gacgaaacag
     2941 cctctacaaa taattttgtt taacgcattt taaaataaaa taaattattt atttaattaa
     3001 acgaatatgt aatgaactgg gatccctcgg taccaaattt tcgaaaaaag acgctgaaaa
     3061 gcgtcttttt tcgttttggt ccgcggccgc cggccgcagg ttgctgtcat aggccggttg
     3121 tccggtagtc cggatgattt aaggaagatt tctcttcttt tttcactttc tccgaacaac
     3181 ctccacctta acttaattat ctttgcagta gctgtcaccg gatgtgcttt ccggtctgat
     3241 gagtccgtga ggacgaaaca gcctctacaa ataattttgt ttaacgcatt ttaaaataaa
     3301 ataaattatt tatttaatta aacgaatatg gtttttactc tggaagattt tgttggcgat
     3361 tggcgtcaga ccgcgggtta taatttggat caagtcctgg aacagggtgg cgtaagctct
     3421 ctgttccaga acctgggtgt gagcgtgacg ccgattcagc gcatcgttct gtccggcgag
     3481 aacggtctga aaattgatat tcatgtgatc atcccgtacg aaggcctgag cggtgaccaa
     3541 atgggtcaaa tcgagaaaat ctttaaagtc gtctacccag ttgacgatca ccacttcaag
     3601 gttatcttgc attacggtac gctggtgatt gatggtgtga ccccgaatat gattgactat
     3661 ttcggccgtc cgtatgaagg cattgccgtt tttgacggta aaaagatcac cgtcaccggt
     3721 accctgtgga atggcaataa gattattgac gagcgtctga ttaacccgga cggcagcctg
     3781 ctgttccgcg tgaccatcaa cggtgtcacg ggttggcgtc tgtgcgagcg catcctggca
     3841 taatgaactg ggatccctcg gtaccaaatt ttcgaaaaaa gacgctgaaa agcgtctttt
     3901 ttcgttttgg tccgcggccg ccggccgcaa tgtgatctgg aagaagcaat gaaagctgct
     3961 gttaagtctc cgaatcaggt attgttcctg acaggtgtat tcccatccgg taaacgcgga
     4021 tactttgcag ttgatctgac tcaggaataa attataaatt aaggtaagaa gattgtagga
     4081 taagctaatg aaatagaaaa aggatgccgt cacacaactt gtcggcattc ttttttgttt
     4141 tattagttga aaatatagtg aaaaagttgc ctaaatatgt atgttaacaa attatttgtc
     4201 gtaactttgc actccaaatc tgtttttaac atatggcact aatgcagaaa aaattgactc
     4261 gttctcagca gaaacacttg gacatcatca acgctgctaa agaagaattc atcgaattcg
     4321 gtttcttggc tgctaacatg gaccgtatca cttcttctgc tgaagtatct aaacgtactt
     4381 tgtaccgtca cttcgaatct aaagaagtat tgttcgaatc tgtattgact atcatcaacg
     4441 actctgtaaa cgaatctatc tcttaccact tcgacccgaa caaatctact gaagaacagt
     4501 tgactgaaat cgcttacaaa gaaatcgacg tattgtacaa aacttacggt atcgctttgg
     4561 ctcgtactat cgtaatggaa ttcttgcgtc agccggaaat ggctaaaact ttgatccaga
     4621 acatctactc tatccgtgct atcactcagt ggttccgttc tgctatcgaa gctaaacgtt
     4681 tgaaagacgc tgacccgaaa ttgatgactg acgtatacgt atctttgttc cagggtttgt
     4741 tcttctggcc gcaggtaatg cacttggact tggaaccgca cggtgaagaa ttgtctcaga
     4801 aaatcgaaac tttgactact atcttcttgc agtcttacgg tgtagctgaa taacaatgaa
     4861 tgggctttcc tcggtaccaa attccagaaa agaggcctcc cgaaaggggg gccttttttc
     4921 gttttggtcc ctaattgcct atcttccagt gatggaacag catttgtgca ttggctgcaa
     4981 caatcagcct tacttgtgcc tgttctattt ccgaaccgac cgcttgtatg aatgcatcaa
     5041 aattcgtttt ctctacgttg gattccttgt tgctcatatt gtgatgataa tttctacaaa
     5101 tatagtcatt ggtaactatc tatgaaactg tttgatactt ttatcagtcc agtagtttta
     5161 caaggtcttt cttcatttct tcgtcaatat ccctgtatcg tctgaaagct ttgcttccct
     5221 ccttgtgtcc cgacagtgcg gaaacaaggt tcgggtcttt cactttttta tagatattgc
     5281 cgataaacgt acgtcttgcc agatggctgc ttgccacttc ataaataggt cgtttgattt
     5341 cgttgtgcgt caacgggtct aagattgtta cgatgcggtc aactccagct aatttgaata
     5401 tcttttttat ggcatcattg tacttttgct cggatatgaa cggcaacagt tttccctcat
     5461 attctttgta gcgttcaagg atttctttcg ctttgtcgtt aagtggaaca cgtaccgtaa
     5521 ccggattccc ctctttggtt ttcttgggaa tatattctat ggcttcattg accacattta
     5581 gtttggtcat tcggtacagg tcgctcaccc tgcatcctat cagtgtctga aatatgaata
     5641 tatccctctg tattgccagt tgtggggtgg cagaaaggtc tgcattaaaa atcctgtccc
     5701 tttcttcgag tgttatataa taaggtgtac catatgtaca ctcctctatc ggaaacttgt
     5761 cgaaaggtct gtttgtggtg cgtttgttat cgaagcacca caggaagaat gtgcgtattc
     5821 ttgaaaaaca gtctatcagc gtgtttttgc ttctgggctg tggtgtcctc ttttcgggaa
     5881 tggcttcata aatgctcggg taaagttcat aatactggta ttcgttctga aagaaatccc
     5941 acatatcccg aagcgtgtca ggtgttacca aatccacatc aaggataaag cccttttgtc
     6001 ctctctttgt agcccttaca tatagttcat aacgcagtaa ggctcttttg acaacccgga
     6061 aatttttctt tcgtacttcc gacaaagggt gcttgtttag aaattcatcg aatagttctc
     6121 caatggtagg cttgataacc acttcctccg gcaaaaaata tttttcagga tggtaaaatt
     6181 tatcaagtgt tgtttttagc cattctttgt ctattgcttc cttttcttgt tgatatacct
     6241 tctcgatata ggttttcaac tggcgtatct cttcatttat atgggtacgc atttcttcat
     6301 tgcagacagc tttcgttttt acacattcgt ctttatcatc ccatagattg ggattgatgg
     6361 ctaactgagt aggagcaacg gagtctaact gccttccgtt tctaaaacgg acatagattg
     6421 tagccatgga ttctgtatca tatcgtttgg ctgctttttt aatgataaag gttactttca
     6481 tagactttca ggttgaattt tactctgctg caaatataaa tattttcccc agcattttcc
     6541 ccacatctgc taaatatttt gcaattcgat taaacttgga ttaaaattta ataggattat
     6601 aacatattga aatacagtgt aattgtggcg tttttccgca tttttctttt tacccacatt
     6661 ttccccactt taattatttc aaatgcgggg tctgggtaca agaaagaaag ctaagtattt
     6721 gatagttcaa tacttagctt tttcttttgc ttgaattttc cccacatttt ccccacacgt
     6781 gcaaaaaata tagcagtaag tcattatttc ttttggttga acgtagagag tagcgatatt
     6841 aaaagaatcc gatgagaaaa gactaatatt tatctatcca ttcagtttga tttctcagga
     6901 ctttacatcg tcctgaaagt atttgttggt accgaggacg cgtaaacatt tacagttgca
     6961 tgtggcctat tgtttttagc cgttaaatat tttataacta ttaaatagcg atacaaattg
     7021 ttcgaaacta atattgttta tatcatatat tctcgcatgt tttaaagctt tattaaattg
     7081 attttttgta aacagttttt cgtactcttt gttaacccat ttcattacaa aagtttcata
     7141 tttttttctc tctttaaatg ccatttttgc tggctttctt tttaatacaa ttaatgtgct
     7201 atccacttta ggttttggat ggaaataata cctaggaatt tttgctaata tagaaatatc
     7261 tacctctgcc attaacagca atgctagtga tctgtttgta tctaataaca ttttagcaaa
     7321 accatattcc actattaaat aacttattgt ggctgaactt tcaaaaacaa tttttcgaat
     7381 tatatttgtg cttatgttgt aaggtatgct gccaaatatt ttatatggat tgtggctagg
     7441 aaatgtaaat ttcagtatat catcatttac tatttgatag ttaggataat ttaagagctt
     7501 attacgagtt acctcacata atttagaatc aatttctatc gccgttacaa aattacatct
     7561 ctttaccaat ccagcagtaa aatgaccttt ccctgcacct atttcaaaga tgttatcttt
     7621 ttcatctaaa cttatgcaat tcattatttt ttctatgtga tattttgaag taataaaatt
     7681 ttgactatct tttatattta ctttgttcat tataacctct ccttaattta ttgcatctct
     7741 tttcgaatat ttatgttttt tgagaaaaga acgtactcat ggttcatccc gatatgcgta
     7801 tcggtctgta tatcagcaac tttctatgtg tttcaactac aatagtcatc tattctcatc
     7861 tttctgagtc caccccctgc aaagcccctc tttacgacat aaaaattcgg tcggaaaagg
     7921 tatgcaaaag atgtttctct ctttaagaga aactcttcgg gatgcaaaaa tatgaaaata
     7981 actccaattc accaaattat atagcgactt ttttacaaaa tgctaaaatt tgttgatttc
     8041 cgtcaagcaa ttgttgagca aaaatgtctt ttacgataaa atgatacctc aatatcaact
     8101 gtttagcaaa acgatatttc tcttaaagag agaaacacct ttttgttcac caatccccga
     8161 cttttaatcc cgcggccatg attgaaaaag gaagagtatg agtattcaac atttccgtgt
     8221 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     8281 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     8341 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     8401 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     8461 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     8521 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     8581 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     8641 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     8701 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     8761 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     8821 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     8881 tattgctgat aaatctggag ccggtgagcg tgggtcacgc ggtatcattg cagcactggg
     8941 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     9001 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     9061 gtcagaccaa gtttactcat aacgcgtcaa ttcgaggggg atcaattccg tgataggtgg
     9121 gctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat ctgttacgcc
     9181 ggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
     9241 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg
     9301 ctgacgccgt tggatacacc aaggaaagtc tacacga
//
