LOCUS       sequence-362496-        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: NTERY02
FEATURES             Location/Qualifiers
     source          1..6391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(82..101)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     292..313
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(542..1357)
                     /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(542..1357)
                     /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     1265..1284
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1349..1368
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1479..2067
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1968..1987
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2221..2238
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2253..2392
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2318..2337)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2478..2500
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2494..2685)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DEFYRSCLARFGDDGENL"
     CDS             complement(3494..4576)
                     /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     4538..4557
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4577..4654)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4860..4879
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4967..4986
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4967..4985
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4990..5006
                     /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             5025..5047
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5664..5693
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(6288..6306)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6302..6349
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(6386..6391,1..450)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
       61 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      121 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      181 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
      241 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      301 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
      361 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
      421 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
      481 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
      541 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
      601 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
      661 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
      721 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
      781 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
      841 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
      901 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
      961 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     1021 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     1081 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     1141 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     1201 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     1261 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     1321 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     1381 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1441 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1501 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1561 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1621 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1681 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1741 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1801 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     1861 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     1921 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     1981 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2041 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2101 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2161 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2221 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     2281 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     2341 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     2401 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     2461 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     2521 acgcgcgagg cagctgcggt aaaactcatc agcgtggtcg tgaagcgatt cacagatgtc
     2581 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     2641 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     2701 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     2761 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     2821 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     2881 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     2941 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     3001 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     3061 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     3121 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     3181 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     3241 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     3301 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     3361 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     3421 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     3481 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3541 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     3601 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     3661 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     3721 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg
     3781 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     3841 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     3901 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     3961 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     4021 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     4081 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     4141 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     4201 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     4261 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     4321 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     4381 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     4441 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     4501 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     4561 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     4621 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     4681 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     4741 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     4801 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     4861 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     4921 atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg cgaaattaat acgactcact
     4981 atagggaaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
     5041 aaggagatat acatatgact atgacaagac tgaagatttc gaaaactctg ctggctgtaa
     5101 tgttgacctc tgccgtcgcg accggctctg cctacgcgga aaacaacgcg cagactacca
     5161 atgaaagcgc agggcaaaaa gtcgatagct ctatgaataa agtcggtaat ttcatggatg
     5221 acagcgccat caccgcgaaa gtgaaggcgg ccctggtgga tcatgacaac atcaagagca
     5281 ccgatatctc tgtaaaaacc gatcaaaaag tcgtgaccct gagcggtttc gttgaaagcc
     5341 aggcccaggc cgaagaggca gtgaaagtgg cgaaaggcgt tgaaggggtg acctctgtca
     5401 gcgacaaact gcacgttcgc gacgctaaag aaggctcggt gaagggctac gcgggtgaca
     5461 ccgccaccac cagtgaaatc aaagccaaac tgctggcgga cgatatcgtc ccttcccgtc
     5521 atgtgaaagt tgaaaccacc gacggcgtgg ttcagctctc cggtaccgtc gattctcagg
     5581 cacaaagtga ccgtgctgaa agtatcgcca aagcggtaga tggtgtgaaa agcgttaaaa
     5641 atgatctgaa aactaagatg ggtcaccacc accaccacca ccaccaccac cacggtagcc
     5701 tgcaagattc ggaagtcaac caagaagcaa agccggaagt caagccggaa gtgaaaccgg
     5761 aaacccatat taacctgaaa gttagtgacg gcagctctga aattttcttt aagatcaaaa
     5821 agaccacgcc gctgcgtcgc ctgatggaag cgtttgccaa acgtcagggc aaggaaatgg
     5881 atagcctgcg tttcctgtat gacggtattc gcatccaggc agatcaagcg ccggaagacc
     5941 tggacatgga agacaacgac atcattgaag cccaccgtga acagattggt ggttattact
     6001 atagcggcgg gagcggaggg tctgggtcta gcggggatgg tgggtcgtct ggggggagcg
     6061 gcggttcggg ttcatctggt gacggcggtt cctcgggtgg ttccggggga gatggttctt
     6121 ccggagatgg cgggagtgat ggggactctg atggttcgga tggtgacggg gacagtgatg
     6181 gtgatgacgg aggggacgac gaggatgacg gttcagacga ttaatgaggc ggctcgagga
     6241 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     6301 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     6361 aactatatcc ggattggcga atgggacgcg c
//
