LOCUS       sequence-365935-        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: NTERY04
FEATURES             Location/Qualifiers
     source          1..6391
                     /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
                     DEFYRSCLARFGDDGENL"
     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
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     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     3489..3508
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3489..3507
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3512..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)."
     RBS             3547..3569
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4186..4215
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(4810..4828)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4824..4871
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4908..5363
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4995..5014)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5205..5226
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5455..6270)
                     /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(5455..6270)
                     /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     6178..6197
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6262..6281
                     /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 atactgtyct 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 gtaaaactca 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 ccgagatgtc 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 ctaccatcga 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 atcgagatcg atctcgatcc
     3481 cgcgaaatta atacgactca ctatagggaa attgtgagcg gataacaatt cccctctaga
     3541 aataattttg tttaacttta agaaggagat atacatatga ctatgacaag actgaagatt
     3601 tcgaaaactc tgctggctgt aatgttgacc tctgccgtcg cgaccggctc tgcctacgcg
     3661 gaaaacaacg cgcagactac caatgaaagc gcagggcaaa aagtcgatag ctctatgaat
     3721 aaagtcggta atttcatgga tgacagcgcc atcaccgcga aagtgaaggc ggccctggtg
     3781 gatcatgaca acatcaagag caccgatatc tctgtaaaaa ccgatcaaaa agtcgtgacc
     3841 ctgagcggtt tcgttgaaag ccaggcccag gccgaagagg cagtgaaagt ggcgaaaggc
     3901 gttgaagggg tgacctctgt cagcgacaaa ctgcacgttc gcgacgctaa agaaggctcg
     3961 gtgaagggct acgcgggtga caccgccacc accagtgaaa tcaaagccaa actgctggcg
     4021 gacgatatcg tcccttcccg tcatgtgaaa gttgaaacca ccgacggcgt ggttcagctc
     4081 tccggtaccg tcgattctca ggcacaaagt gaccgtgctg aaagtatcgc caaagcggta
     4141 gatggtgtga aaagcgttaa aaatgatctg aaaactaaga tgggtcacca ccaccaccac
     4201 caccaccacc accacggtag cctgcaagat tcggaagtca accaagaagc aaagccggaa
     4261 gtcaagccgg aagtgaaacc ggaaacccat attaacctga aagttagtga cggcagctct
     4321 gaaattttct ttaagatcaa aaagaccacg ccgctgcgtc gcctgatgga agcgtttgcc
     4381 aaacgtcagg gcaaggaaat ggatagcctg cgtttcctgt atgacggtat tcgcatccag
     4441 gcagatcaag cgccggaaga cctggacatg gaagacaacg acatcattga agcccaccgt
     4501 gaacagattg gtggtggtgg ttcgagctat tactatggag ggtctgggtc tagcggggat
     4561 ggtgggtcgt ctggggggag cggcggttcg ggttcatctg gtgacggcgg ttcctcgggt
     4621 ggttccgggg gagatggttc ttccggagat ggcgggagtg atggggactc tgatggttcg
     4681 gatggtgacg gggacagtga tggtgatgac ggaggggacg acgaggatga cggttcagac
     4741 gattaatgag gcggctcgag gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     4801 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     4861 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     4921 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4981 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5041 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5101 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     5161 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5221 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     5281 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     5341 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     5401 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     5461 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     5521 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     5581 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     5641 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     5701 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     5761 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     5821 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     5881 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     5941 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     6001 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     6061 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     6121 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     6181 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     6241 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     6301 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6361 cagaccccgt agaaaagatc aaaggatctt c
//
