LOCUS       sequence-416764-        6513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of recombinant calcium sensor jGCaMP8f F366H high dynamic
            range variant in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6513)
  AUTHORS   Tran O, Hughes HJ, Carter T, Torok K
  TITLE     Development and characterization of novel jGCaMP8f calcium sensor 
            variants with improved kinetics and fluorescence response range.
  JOURNAL   Front Cell Neurosci. 2023 May 18;17:1155406. doi: 
            10.3389/fncel.2023.1155406. eCollection 2023.
  PUBMED    37275778
REFERENCE   2  (bases 1 to 6513)
  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: "Front Cell 
            Neurosci."; date: "2023-05-18"; volume: "17:1155406. doi"; pages: " 
            10.3389/fncel.2023.1155406. eCollection 2023"
COMMENT     Sequence Label: pET30b jGCaMP8f F366H
FEATURES             Location/Qualifiers
     source          1..6513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     154..171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    186..325
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(251..270)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     411..433
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(427..618)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1427..2509)
                     /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     2471..2490
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2510..2587)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2793..2812
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2900..2919
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2900..2918
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2919..2943
                     /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             2958..2980
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2991..3008
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     3290..3311
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3417..3438)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3678..3697)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             4273..4290
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4343..4361)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4357..4404
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4441..4896
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4528..4547)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4738..4759
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4988..5803)
                     /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(4988..5803)
                     /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     5711..5730
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5795..5814
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5925..6513
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6414..6433
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      121 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      181 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
      241 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
      301 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
      361 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
      421 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
      481 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
      541 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
      601 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
      661 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
      721 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
      781 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
      841 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
      901 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
      961 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     1021 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     1081 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     1141 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     1201 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     1261 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     1321 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     1381 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     1441 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     1501 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     1561 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     1621 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     1681 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     1741 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     1801 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     1861 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     1921 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     1981 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     2041 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     2101 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     2161 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     2221 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     2281 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     2341 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     2401 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     2461 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     2521 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     2581 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     2641 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     2701 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     2761 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     2821 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     2881 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     2941 tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg catcatcacc
     3001 atcatcacac gcgtcgcaag aagaccttca aggaggtggc caccgccgtg aagatcatcg
     3061 cccgcctgat gggcctgaag atcaacgtct atatcaaggc cgacaagcag aagaacggca
     3121 tcaaggcgaa cttccacatc cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc
     3181 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     3241 tgagcgtgga gtccaaactt tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     3301 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcggta
     3361 ccggagggag catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     3421 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggtgagggcg
     3481 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     3541 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     3601 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac atccaggagc
     3661 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     3721 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     3781 tcctggggca caagctggag tacaacctgc cggaccaact gactgaagag cagatcgcag
     3841 aatttaaaga ggctttctcc ctatttgaca aggacgggga tgggacaata acaaccaagg
     3901 agctggggac ggtgatgcgg tctctcggac tgaaccccac agaagcagag ctgcaggaca
     3961 tgatcaatga agtagatgcc gacggtgacg gcacaatcga cttccctgag ttcctgacaa
     4021 tgatggcaag aaaaatgaaa tacagggaca cggaagaaga aattagagaa gcgttcggtg
     4081 tgcatgataa ggatggcaat ggctacatca gtgcagcaga gcttcgccac gtgatgacaa
     4141 accttggaga gaagttaaca gatgaagagg ttgatgaaat gatcagggaa gcagacatcg
     4201 atggggatgg tcaggtaaac tacgaagagt ttgtacaaat gatgacagcg aagtgatgcg
     4261 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     4321 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     4381 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     4441 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4501 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4561 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4621 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     4681 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4741 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     4801 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     4861 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     4921 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     4981 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     5041 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     5101 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     5161 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     5221 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     5281 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     5341 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     5401 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     5461 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     5521 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     5581 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     5641 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     5701 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     5761 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     5821 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     5881 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5941 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6001 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6061 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6121 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6181 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6241 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6301 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6361 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6421 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6481 tgatgctcgt caggggggcg gagcctatgg aaa
//
