LOCUS       sequence-365939-        6391 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nanopore-addressable protein reporter for expression in bacterial 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6391)
  AUTHORS   Cardozo N, Zhang K, Doroschak K, Nguyen A, Siddiqui Z, Bogard N, 
            Strauss K, Ceze L, Nivala J
  TITLE     Multiplexed direct detection of barcoded protein reporters on a 
            nanopore array.
  JOURNAL   Nat Biotechnol. 2022 Jan;40(1):42-46. doi: 
            10.1038/s41587-021-01002-6. Epub 2021 Aug 12.
  PUBMED    34385692
REFERENCE   2  (bases 1 to 6391)
  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"; doi: 
            "10.1038/s41587-021-01002-6"; journalName: "Nat Biotechnol"; date: 
            "2022-01"; volume: "40"; issue: "1"; pages: "42-46"
COMMENT     Sequence Label: NTERY03
FEATURES             Location/Qualifiers
     source          1..6391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(600..1682)
                     /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     1644..1663
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1683..1760)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1966..1985
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2073..2092
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2073..2091
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2096..2112
                     /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)."
     RBS             2131..2153
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2770..2799
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(3394..3412)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3408..3455
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3492..3947
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3579..3598)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3789..3810
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4039..4854)
                     /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(4039..4854)
                     /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     4762..4781
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4846..4865
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4976..5564
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5465..5484
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5718..5735
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5750..5889
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5815..5834)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5975..5997
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5991..6182)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DEFYRSCLARFGDDGENL"
ORIGIN
        1 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
       61 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
      121 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
      181 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
      241 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
      301 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
      361 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
      421 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
      481 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
      541 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
      601 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
      661 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
      721 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
      781 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
      841 catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc
      901 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
      961 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     1021 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     1081 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     1141 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     1201 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     1261 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     1321 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     1381 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     1441 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     1501 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     1561 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     1621 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     1681 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     1741 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     1801 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     1861 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     1921 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     1981 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     2041 gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactatag ggaaattgtg
     2101 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
     2161 atgactatga caagactgaa gatttcgaaa actctgctgg ctgtaatgtt gacctctgcc
     2221 gtcgcgaccg gctctgccta cgcggaaaac aacgcgcaga ctaccaatga aagcgcaggg
     2281 caaaaagtcg atagctctat gaataaagtc ggtaatttca tggatgacag cgccatcacc
     2341 gcgaaagtga aggcggccct ggtggatcat gacaacatca agagcaccga tatctctgta
     2401 aaaaccgatc aaaaagtcgt gaccctgagc ggtttcgttg aaagccaggc ccaggccgaa
     2461 gaggcagtga aagtggcgaa aggcgttgaa ggggtgacct ctgtcagcga caaactgcac
     2521 gttcgcgacg ctaaagaagg ctcggtgaag ggctacgcgg gtgacaccgc caccaccagt
     2581 gaaatcaaag ccaaactgct ggcggacgat atcgtccctt cccgtcatgt gaaagttgaa
     2641 accaccgacg gcgtggttca gctctccggt accgtcgatt ctcaggcaca aagtgaccgt
     2701 gctgaaagta tcgccaaagc ggtagatggt gtgaaaagcg ttaaaaatga tctgaaaact
     2761 aagatgggtc accaccacca ccaccaccac caccaccacg gtagcctgca agattcggaa
     2821 gtcaaccaag aagcaaagcc ggaagtcaag ccggaagtga aaccggaaac ccatattaac
     2881 ctgaaagtta gtgacggcag ctctgaaatt ttctttaaga tcaaaaagac cacgccgctg
     2941 cgtcgcctga tggaagcgtt tgccaaacgt cagggcaagg aaatggatag cctgcgtttc
     3001 ctgtatgacg gtattcgcat ccaggcagat caagcgccgg aagacctgga catggaagac
     3061 aacgacatca ttgaagccca ccgtgaacag attggtggtg gtggttatta ctatgggagc
     3121 ggagggtctg ggtctagcgg ggatggtggg tcgtctgggg ggagcggcgg ttcgggttca
     3181 tctggtgacg gcggttcctc gggtggttcc gggggagatg gttcttccgg agatggcggg
     3241 agtgatgggg actctgatgg ttcggatggt gacggggaca gtgatggtga tgacggaggg
     3301 gacgacgagg atgacggttc agacgattaa tgaggcggct cgaggatccg gctgctaaca
     3361 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     3421 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     3481 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3541 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3601 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3661 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3721 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3781 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3841 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3901 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     3961 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4021 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     4081 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     4141 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     4201 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     4261 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     4321 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     4381 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     4441 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     4501 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     4561 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     4621 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     4681 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     4741 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     4801 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     4861 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     4921 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4981 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5041 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5101 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     5161 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5221 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5281 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5341 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5401 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5461 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5521 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5581 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5641 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5701 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     5761 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     5821 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     5881 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5941 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     6001 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa actcatcagc gtggtcgtga
     6061 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     6121 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     6181 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     6241 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     6301 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     6361 tgccagcgct tcgttaatac agatgtaggt g
//
