LOCUS       sequence-433255-        5372 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Plasmid with cleavable His-tag for expression of the HyPer-FLEX 
            sensor for hydrogen peroxide in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5372)
  TITLE     HyPerFAST - a genetically encoded fluorescent sensor for hydrogen 
            peroxide with tunable colors for in vivo imaging
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5372)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pD441-H6-SUMO-HyPerFLEX
FEATURES             Location/Qualifiers
     source          1..5372
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      166..754
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     655..674
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     908..925
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1023..1040
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1024..1040
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1497..1513
                     /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)."
     promoter        join(1538..1552,1553..1558,1559..1575,1576..1582)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1558..1574
                     /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)."
     protein_bind    1588..1607
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     RBS             1648..1656
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             1674..1691
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1698..1991
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(3300..3319)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        3386..3477
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3478..4287
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             join(4370..5372,1..80)
                     /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     complement(4389..4408)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
       61 gactggaaag cgggcagtga ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc
      121 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
      181 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
      241 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
      301 accaaatact gttcttctag tgtagccgta gttagcccac cacttcaaga actctgtagc
      361 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
      421 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
      481 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
      541 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
      601 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
      661 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
      721 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
      781 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
      841 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
      901 cgagcgcagc gagtcagtga gcgaggaagc ggaaggcgag agtagggaac tgccaggcat
      961 caaactaagc agaaggcccc tgacggatgg cctttttgcg tttctacaaa ctctttctgt
     1021 gttgtaaaac gacggccagt cttaagctcg ggccccctgg gcggttctga taacgagtaa
     1081 tcgttaatcc gcaaataacg taaaaacccg cttcggcggg tttttttatg gggggagttt
     1141 agggaaagag catttgtcag aatatttaag ggcgcctgtc actttgcttg atatatgaga
     1201 attatttaac cttataaatg agaaaaaagc aacgcacttt aaataagata cgttgctttt
     1261 tcgattgatg aacacctata attaaactat tcatctatta tttatgattt tttgtatata
     1321 caatatttct agtttgttaa agagaattaa gaaaataaat ctcgaaaata ataaagggaa
     1381 aatcagtttt tgatatcaaa attatacatg tcaacgataa tacaaaatat aatacaaact
     1441 ataagatgtt atcagtattt attatgcatt tagaataaat tttgtgtcgc ccttaattgt
     1501 gagcggataa caattacgag cttcatgcac agtgaaatca tgaaaaattt atttgctttg
     1561 tgagcggata acaattataa tatgtggaat tgtgagcgct cacaattcca caacggtttc
     1621 cctctagaaa taattttgtt taacttttaa ggaggtaaca tatgggcagc agccatcatc
     1681 atcatcatca cggtaccatg tcggactcag aagtcaatca agaagctaag ccagaggtca
     1741 agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga tcttcagaga
     1801 tcttcttcaa gatcaaaaag accactcctt taagaaggct gatggaagcg ttcgctaaaa
     1861 gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga attcaagctg
     1921 atcagacccc tgaagatttg gacatggagg ataacgatat tattgaggct cacagagaac
     1981 agattggtgg tatgcacctg gctaatgagg agcaaaacga attggaaggt gcttttaagc
     2041 tgggcttgat ctttaccgtc gccccgtact tgctgcctaa gttgatcgtc agcctgcgcc
     2101 gcaccgcccc gaagatgcct ttgatgttgg aggagaatta cacccacacc cttaccgagt
     2161 cactgaaacg cggcgacgtt gacgccatca ttgtggcaga gccgtttcaa gagccgggta
     2221 ttgtgactga acctttgtat gacgagccgt ttttcgttat cgttccgaag ggccatagct
     2281 tcgaggagtt ggatgccgtt agccctcgca tgttgggcga agaacaagtt ctgcttctga
     2341 ccgagggcaa ctgtatgcgt gaccaggtgc ttagcatttt taccaaggtc aaggtgcaca
     2401 tgaagaaagc cctttccggt gacagctatt gggtctttgt gaaacgggta ggtggcactg
     2461 gcggtgagca tgttgtcttt ggcagtgagg acatcgagaa cactatggcc aaaatggacg
     2521 acggacaact ggatgggttg gcctttggcg caattcagct cgatggtgac gggaatatcc
     2581 tgcagtacaa tgctgctgaa ggagacatca caggcagaga tcccaaacag gtgattggga
     2641 agaacttctt caaggatgtt gcacctggaa cggattctcc cgagttttac ggcaaattca
     2701 aggaaggcgt agcgtcaggg aatctgaaca ccatgttcga atggatgata ccgactctta
     2761 ccgatcatag ttgcagcgag cttgccgcca aacagcgcat tcagggtctc accaacaccc
     2821 ttcaaggctc aagcatcgat accatccgcc acatggttgc cagtggcttg gcaatcagcg
     2881 tcttgcctgc cactgccctt accgaaaacg accacatgct gtttagcatt atcccgtttg
     2941 aaggcacccc gcctagccgc cgcgttgtcc tggcctatcg ccgcaacttt gtccgcccta
     3001 aggccctttc agccatgaaa gccgcaatta tgcaaagcca actgcatggt gttagcttca
     3061 tctgcgattg ataactcgag gcggccgccg acaagcttgg ttgaccccaa gggcgacacc
     3121 ccctaattag cccgggcgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg
     3181 cctggcagtt ccctactctc gcatggggag tccccacact accatcggcg ctacggcgtt
     3241 tcacttctga gttcggcatg gggtcaggtg ggaccaccgc gctactgccg ccaggcaaac
     3301 aaggggtgtt atgagccata ttcaggtata aatgggctcg cgataatgtt cagaattggt
     3361 taattggttg taacactgac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3421 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagaatatg
     3481 agccatattc aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta
     3541 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg
     3601 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     3661 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc acttccgacc
     3721 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga
     3781 aaaacagcgt tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     3841 ctggcagtgt tcctgcgccg gttgcactcg attcctgttt gtaattgtcc ttttaacagc
     3901 gatcgcgtat ttcgcctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     3961 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     4021 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     4081 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     4141 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     4201 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcaattt
     4261 catttgatgc tcgatgagtt tttctaagcg gcgcgccatc gaatggcgca aaacctttcg
     4321 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaata tgaaaccagt
     4381 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     4441 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     4501 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     4561 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     4621 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     4681 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     4741 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     4801 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     4861 ggacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     4921 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     4981 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     5041 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     5101 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     5161 tggtgcggat atctcggtag tgggatacga cgataccgaa gatagctcat gttatatccc
     5221 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     5281 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgccag tctcactggt
     5341 gaaaagaaaa accaccctgg cgcccaatac gc
//
