LOCUS       sequence-207312-        7179 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression plasmid for potassium ion ratiometric indicator
            KIRIN1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7179)
  AUTHORS   Shen Y, Wu SY, Rancic V, Aggarwal A, Qian Y, Miyashita SI, Ballanyi 
            K, Campbell RE, Dong M
  TITLE     Genetically encoded fluorescent indicators for imaging intracellular
            potassium ion concentration.
  JOURNAL   Commun Biol. 2019 Jan 14;2:18. doi: 10.1038/s42003-018-0269-2. 
            eCollection 2019.
  PUBMED    30652129
REFERENCE   2  (bases 1 to 7179)
  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"; journalName: "Commun 
            Biol."; date: "2019-01-14"; volume: "2:18. doi"; pages: " 
            10.1038/s42003-018-0269-2. eCollection 2019"
COMMENT     Sequence Label: pET28a-KIRIN1
FEATURES             Location/Qualifiers
     source          1..7179
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     50..69
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        50..68
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    69..93
                     /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             108..130
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             149..166
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             176..193
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(251..272)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(512..531)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2093..2110
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2163..2181)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2177..2224
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2261..2716
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2348..2367)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2558..2579
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2808..3623)
                     /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(2808..3623)
                     /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     3531..3550
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3615..3634
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3745..4333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4234..4253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4487..4504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4519..4658
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4584..4603)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4744..4766
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4760..4951)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5760..6842)
                     /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     6804..6823
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6843..6920)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7126..7145
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
       61 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
      121 aagaaggaga tataccatgg gcagcagcca tcatcatcat catcacagca gcggcctggt
      181 gccgcgcggc agccatatgg ctagcatggt gagcaagggc gaggagctgt tcaccggggt
      241 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
      301 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
      361 caagctgccc gtgccctggc ccaccctcgt gaccaccctg agctggggcg tgcagtgctt
      421 cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
      481 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
      541 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
      601 ggaggacggc aacatcctgg ggcacaagct ggagtacaac gccatccacg gcaacgtcta
      661 tatcaccgcc gacaagcaga agaacggcat caaggccaac ttcggcctca actgcaacat
      721 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
      781 ccccgtgctg ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc
      841 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgcggccg caggcctgtt
      901 caacttcgtg aaggacgccg gcgagaagct gtgggatgcc gtgacaggcc agcacgacaa
      961 ggacgaccag gccaagaagg tgcaggaaca cctgaacaag accggcatcc ccgacgccga
     1021 caaagtgaac atccagatcg ccgacggcaa ggccacagtg acaggcgacg gactgagcca
     1081 ggaagccaaa gaaaagatcc tggtggccgt gggcaacatc agcggaatcg ccagcgtgga
     1141 cgatcaagtg aaaaccgcca cccctgccac cgccagccag ttctacacag tgaagtccgg
     1201 cgacaccctg agcgccatca gcaaacaggt gtacggcaac gccaacctgt acaacaagat
     1261 tttcgaggcc aacaagccca tgctgaagtc ccccgacaaa atctaccccg gccaggtgct
     1321 gagaatcccc gaggaactgg agctcatggg tggggtccag cttgcagatc actaccagca
     1381 gaatactccg atcggggatg ggccggttct cctgcctgac aaccactacc tgtcatacca
     1441 gtccaagctg tccaaagatc ctaatgaaaa gcgggatcac atggtcctgc tggaatttgt
     1501 caccgccgcc ggaatcactc tcggaatgga cgagctgtac aaaggtggga ccgggggatc
     1561 tatggtgagc aagggcgaag aattgttcac tggtgtagtc cctattttgg tggaactgga
     1621 tggggatgtc aacggtcata aattcagcgt gcgaggggag ggtgaggggg acgcaactaa
     1681 tggcaaattg acactcaagc tcatctgcac aacggggaaa ctgcccgtgc cgtggcccac
     1741 actcgttact actctaggat atggccttat gtgtttcgcc cgctacccag accacatgaa
     1801 acaacacgat ttctttaaat cagccatgcc agaggggtac gtccaggaga gaactatctt
     1861 cttcaaggac gatggtaatt acaagactag ggccgaggtg aaattcgaag gggatacact
     1921 tgtcaaccga atcgagctga agggaatcga ctttaaggag gacggaaaca tactcgggca
     1981 taagttggaa tacaactaca acagtcacaa tgtttacatc acagccgata agcaaaagaa
     2041 cggcattaag gctaatttta agatccgtca caacattgag gattaactcg agcaccacca
     2101 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     2161 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     2221 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     2281 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2341 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2401 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2461 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2521 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2581 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2641 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     2701 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2761 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     2821 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     2881 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     2941 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     3001 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     3061 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     3121 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     3181 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     3241 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     3301 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     3361 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     3421 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     3481 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     3541 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     3601 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     3661 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3721 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3781 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3841 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3901 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3961 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4021 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4081 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4141 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4201 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4261 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4321 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4381 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4441 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4501 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4561 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     4621 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4681 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4741 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4801 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     4861 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     4921 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     4981 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     5041 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     5101 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     5161 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     5221 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     5281 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     5341 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     5401 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     5461 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     5521 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     5581 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     5641 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     5701 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     5761 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5821 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     5881 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     5941 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     6001 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     6061 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     6121 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     6181 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     6241 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     6301 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     6361 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     6421 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     6481 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     6541 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     6601 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     6661 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     6721 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     6781 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     6841 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     6901 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     6961 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     7021 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     7081 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     7141 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgcc
//
