LOCUS       sequence-362500-        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: NTERY06
FEATURES             Location/Qualifiers
     source          1..6391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      14..469
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(101..120)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     311..332
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(561..1376)
                     /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(561..1376)
                     /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     1284..1303
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1368..1387
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1498..2086
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1987..2006
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2240..2257
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2272..2411
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2337..2356)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2497..2519
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2513..2704)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DEFYRSCLARFGDDGENL"
     CDS             complement(3513..4595)
                     /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     4557..4576
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4596..4673)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4879..4898
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4986..5005
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4986..5004
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5009..5025
                     /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             5044..5066
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5683..5712
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(6307..6325)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6321..6368
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
       61 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
      121 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
      181 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
      241 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
      301 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
      361 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
      421 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
      481 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
      541 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
      601 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
      661 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
      721 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
      781 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
      841 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
      901 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
      961 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     1021 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     1081 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     1141 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     1201 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     1261 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     1321 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     1381 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     1441 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1501 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1561 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1621 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     1681 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1741 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1801 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1861 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     1921 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     1981 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2041 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2101 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2161 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2221 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     2281 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     2341 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     2401 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     2461 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     2521 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aaactcatca gcgtggtcgt
     2581 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     2641 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     2701 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     2761 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     2821 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     2881 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     2941 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     3001 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     3061 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     3121 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     3181 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     3241 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     3301 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     3361 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     3421 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     3481 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3541 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3601 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     3661 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     3721 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     3781 cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     3841 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     3901 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     3961 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     4021 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     4081 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     4141 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     4201 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     4261 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     4321 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     4381 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     4441 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     4501 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     4561 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     4621 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     4681 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     4741 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     4801 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     4861 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     4921 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc
     4981 gaaattaata cgactcacta tagggaaatt gtgagcggat aacaattccc ctctagaaat
     5041 aattttgttt aactttaaga aggagatata catatgacta tgacaagact gaagatttcg
     5101 aaaactctgc tggctgtaat gttgacctct gccgtcgcga ccggctctgc ctacgcggaa
     5161 aacaacgcgc agactaccaa tgaaagcgca gggcaaaaag tcgatagctc tatgaataaa
     5221 gtcggtaatt tcatggatga cagcgccatc accgcgaaag tgaaggcggc cctggtggat
     5281 catgacaaca tcaagagcac cgatatctct gtaaaaaccg atcaaaaagt cgtgaccctg
     5341 agcggtttcg ttgaaagcca ggcccaggcc gaagaggcag tgaaagtggc gaaaggcgtt
     5401 gaaggggtga cctctgtcag cgacaaactg cacgttcgcg acgctaaaga aggctcggtg
     5461 aagggctacg cgggtgacac cgccaccacc agtgaaatca aagccaaact gctggcggac
     5521 gatatcgtcc cttcccgtca tgtgaaagtt gaaaccaccg acggcgtggt tcagctctcc
     5581 ggtaccgtcg attctcaggc acaaagtgac cgtgctgaaa gtatcgccaa agcggtagat
     5641 ggtgtgaaaa gcgttaaaaa tgatctgaaa actaagatgg gtcaccacca ccaccaccac
     5701 caccaccacc acggtagcct gcaagattcg gaagtcaacc aagaagcaaa gccggaagtc
     5761 aagccggaag tgaaaccgga aacccatatt aacctgaaag ttagtgacgg cagctctgaa
     5821 attttcttta agatcaaaaa gaccacgccg ctgcgtcgcc tgatggaagc gtttgccaaa
     5881 cgtcagggca aggaaatgga tagcctgcgt ttcctgtatg acggtattcg catccaggca
     5941 gatcaagcgc cggaagacct ggacatggaa gacaacgaca tcattgaagc ccaccgtgaa
     6001 cagattggtg gtggtggttc gagcggcggg agcggatatt actattctag cggggatggt
     6061 gggtcgtctg gggggagcgg cggttcgggt tcatctggtg acggcggttc ctcgggtggt
     6121 tccgggggag atggttcttc cggagatggc gggagtgatg gggactctga tggttcggat
     6181 ggtgacgggg acagtgatgg tgatgacgga ggggacgacg aggatgacgg ttcagacgat
     6241 taatgaggcg gctcgaggat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     6301 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     6361 gttttttgct gaaaggagga actatatccg g
//
