LOCUS       sequence-491309-        7945 bp    DNA     circular SYN 31-OCT-2025
DEFINITION  Polyubiquitin promoter driving expression of cytoplasmic hydrogen 
            peroxide biosensor roGFP2-ORP1. Flanked by ITRs compatible with 
            minos-mediated transgenesis..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7945)
  TITLE     Redox biosensor expression plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7945)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 31, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: LvPolyubiquitin-roGFP2-ORP1-minos-lox-ITR
FEATURES             Location/Qualifiers
     source          1..7945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     742..764
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     761..777
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     761..777
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    complement(1174..1207)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             join(4682..4684,4685..4687,4688..5398)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFISTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTCSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4727..4748)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4988..5007)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5335..5356
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6024..6158
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6048..6067)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6102..6121
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(6182..6215)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(6610..6627)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6610..6626)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6619..6641)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        6740..6844
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6845..6913,6914..7705)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7063..7082)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     protein_bind    7805..7829
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttccagc
      661 gacatatgcc atacgccgaa ctgaactcca cctaatgtct tggagattgt cggatagcgc
      721 ggaataggat tgtgccctct gagcggataa caatttcaca caggaaacag ctatgaccat
      781 gacacatgca tccaccatcg cagacttatc atcactgagc ctccacctag cctcattctc
      841 tatgctagct taagatattt ttcgttttct acgagcccca accactatta attcgaacag
      901 catgtttttt ttgcagtgcg caatgtttaa cacactatat tatcaatact actaaagata
      961 acacatacca atgcatttcg tctcaaagag aattttattc tcttcacgac gaaaaaaaaa
     1021 gttttgctct atttccaaca acaacaaaaa tatgagtaat ttattcaaac ggtttgctta
     1081 agagataaga aaaaagtgac cactattaat tcgaacgcgg cgtaagctac cttaatctca
     1141 agaagagcaa aacaaaagca actaatgtaa cggataactt cgtatagcat acattatacg
     1201 aagttataac catatgtgac gtctacggat ccgcatgcac tagtcctcac aacatttctt
     1261 cccttttttg cgggagttgg ggcaaataat cctgacaatg tccaatgatt attttaaaaa
     1321 gtgctatgta caatcgcttt gtatttgtat aattgttata aaaaatgcat tgtatgtatt
     1381 tatatttaca tacataatta ttctacaaag tgtattgtac aatcacttta tatcaacttt
     1441 agatgtatcg tgttattagt gtcaaatctg actgcgtgca tactgattcg atgccaaaag
     1501 tggattaatg cctccttgtg ccccgcgtgt ctcttggcaa ctagagcatt attgtagaat
     1561 attggggcac ctaaaataca ctgacaagca atcatccaaa aatatctaac aaaatactaa
     1621 acaataacag gacggtcatt tttctgatat ctaaaacaag tttttgttga agggacattg
     1681 tgcaagactt tcaaaaactt attaagaatg ttgaaccgtt tacttttttt attttaacac
     1741 gtgatactat agtatacata acgattgggc gaaaaaatac cgtgacagtc aatcaaacaa
     1801 tctaaattcc cactttgcag aatcactttt gtgattccct tgtatcttca acaagatata
     1861 gatagatgga tagcatggaa gtttttgttt tttacttaga aatggtaaaa cgatgttttt
     1921 tccccagggt cttttccccc tatgtaaatt atgtttcatt ctttcaataa aaaaaaacac
     1981 aaactgaaaa aaaacttata agcaaaaaaa aaggcgtaaa tattttttta tgaactctgt
     2041 taaacggaaa caacaacaac acagtcgcca atatatacct cccgcgacca gtgatcattc
     2101 cagaaaatgt tttgggccga aattatattg cataattccg aaattgcgag tcatcatcgt
     2161 ctgcaatatc tggcgcgggt taatatgacg tcatgtgcca tagatacaca atctatcttt
     2221 attacccaat agaaaaggat gaaatagtcc atgtgacaca gactgaccaa tcgcaagtca
     2281 acacaaagca ccatataaaa cacacgcaag cgtcgcagtg tcaataaatt gcatgagact
     2341 tccgcagagg ttgcactcga gaagaactcg tccatttttt tccagaaaaa ctctaatttc
     2401 aaagtaagta gacgaggttt tcacattatt ttaaggcagt tatttcaatg aagatgcagt
     2461 ttgtaaatgt ttgatttaaa aaaagcactt ttgaagaaga atgatgatga agaacataaa
     2521 ttgtcaacga ccacatcgtc gtcattattc gtgtgctttt tttttgttgt aatttatgtt
     2581 tggctttcca aatgaaggca atataggaac attaacagca tgaacaaagt ctcatattta
     2641 gtttgattga aagttcaaat tatagggcgt agaaatcatt gaaattgtag ggtggatacc
     2701 gtggattatt gcgtcgtgtg tgtgcatgat aagatcgtct gactcagacg tcgcgtcttt
     2761 tgttgtgtgg tgtgtgtggt atggatgatg atcaattgtg atgtttggtg gggattttca
     2821 tgaacgctga attaaaagac aatgagtgga aatggccatg ccaatttctg gtccatggat
     2881 tgtgtgaatg caatgacaat ttcattcaat ttttgtctgg gctgccatga tgaacatgat
     2941 gaagtctgaa ctgaagtgtt gtgattgaaa tgacaaagaa tttttgtcat gagaaagaat
     3001 gaggcatgta cggtacggta acttaaaacg ttgatagcta ggtgtagatt ttctagatac
     3061 gtttggcagt aggcctagat ctagaggcac ttggccaatc atgaatagtc tatgggagac
     3121 aatttcccat attgaagact tgctttgcaa aagaaacgac accaacaaag cgtaattttt
     3181 tccaccccaa attttttcgc gaaggtcaga acagaagccc atggcaaagg cctgttttgt
     3241 atgtgcatgg ccatggatga aaaaaatata aatacaaaag ttggtgaatt tttaaagatg
     3301 aaaaggggta atttttcatg aggtatctgc ttaggcccta tcacattttt atttgctgcc
     3361 aaggtaattc ttttgctgca tgtgtaaaaa aaggaaattg gtctcccaaa ctgggagtgg
     3421 agactgtctc tgaaattgga tacccaagtt ctttaatctt tacaaaattc tgatattgaa
     3481 aataatctct ctcatgggag atgttctccc atattggctg tgcaccagtt ggtcggtgtg
     3541 caaagtctag acaatacgta tccaggcact atcgcctatg aataatgcaa tagcagtgca
     3601 gttcacaacg gcctctctcc cgatcagcgc agatgttggg gaactaattt caacttttgt
     3661 cgtaatttat gtcaaataac tgacagtcgt aacttaagac cctaatcagc taatgttaat
     3721 ccttgacctg aattgcaggc atctgaaacg gctgtaatcc agcttcgatc ttaatgtcat
     3781 agttcaaatt ctcagtagat ttaactgagc gtggtgcgtg tttggccatt gccatagttt
     3841 ttatatggac tgaagtcaac gaccaaaaga tctgctcaag gcagcccaac tgcattagca
     3901 agattttggt tgctattcag tcaatatcag aactacggca atggacgagt gcggcgatgt
     3961 caacctacat gtaactgaga attcaaacta tgacattgag aacgaagcca aaatacagca
     4021 aatattgatg tggttgaaac ttgggtcaag gatcaaatgg acaatatttg gcaaaaaaga
     4081 taaattttga ggtgaaattg tttcctgaca ttgccatgtt ggttggtgtg aaactgcagc
     4141 gcaagacaat gaaggaagag actttataga gtaagctgcc ccgaatttga catgttttga
     4201 accatttttt gttcttaagt cttttcacat aatgaaaatt gcattaaaat gatcaatgtt
     4261 ggttgaatat atttagaatt gcaatgttga atttctccat tttgaatatt tcataaaaaa
     4321 tctgcactgt accaccatgt tttgctcccg ccacagattt cttaatttta gagcatttta
     4381 tggctacctc atttcttaaa tctgagcaaa gctaccgcat gttattgaaa tatggcacag
     4441 attcgcattt ttaaagggga ttttggtgtt caagtcgtga cttgcatgtg gtgggtgata
     4501 atgcctgtgt atgtcaatgc atgtggttcg tgttgtgtgg attgactgag ctgacttgtg
     4561 gaataagttt gccatgttga attccaggca tgctactctg aataaaggtc tttagaaaaa
     4621 cagattccaa taatgttttc atttttttgt caattttcta caggtcagtg cgatcgcatc
     4681 gatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     4741 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     4801 cggcaagctg accctgaagt tcatctccac caccggcaag ctgcccgtgc cctggcccac
     4861 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     4921 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     4981 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     5041 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     5101 caagctggag tacaactaca actgccacaa cgtctatatc atggccgaca agcagaagaa
     5161 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     5221 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     5281 ctacctgagc acctgctccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     5341 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagac
     5401 tagtggtggt tcaggtggtg gtggttcagg tggtggtggt tcaggtggag gaggatcagg
     5461 aggaggagga tcaggaggag gaggatcagg aggagaattc gatatctcag aattctataa
     5521 gctagcacct gttgacaaga aaggccaacc attccccttc gaccaattaa agggaaaagt
     5581 ggtgcttatc gttaatgttg cctccaaatg tggattcact cctcaataca aagaactaga
     5641 ggccttgtac aaacgttata aggacgaagg atttaccatc atcgggttcc catgcaacca
     5701 gtttggccac caagaacctg gctctgatga agaaattgcc cagttctgcc aactgaacta
     5761 tggcgtgact ttccccatta tgaaaaaaat tgacgttaat ggtggcaatg aggaccctgt
     5821 ttacaagttt ttgaagagcc aaaaatccgg tatgttgggc ttgagaggta tcaaatggaa
     5881 ttttgaaaaa ttcttagtcg ataaaaaggg taaagtgtac gaaagatact cttcactaac
     5941 caaaccttct tcgttgtccg aaaccatcga agaacttttg aaagaggtgg aatagctaga
     6001 actatagtga gtcgtattac gtagatccag acatgataag atacattgat gagtttggac
     6061 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     6121 ctttatttgt aaccattata agctgcaata aacaagttgg ccgccttcac gcgtggttac
     6181 cataacttcg tataggatac tttatacgaa gttatccgcg ggggaaatta attaaattat
     6241 tgttttaagt atgatagtaa atcacattac gccgcgttcg aattaatagt ggtcactttt
     6301 ttcttatctc ttaagcaaac cgtttgaata aattactcat atttttgttg ttgttggaaa
     6361 tagagcaaaa cttttttttt cgtcgtgaag agaataaaat tctctttgag acgaaatgca
     6421 ttggtatgtg ttatctttag tagtattgat aatatagtgt gttaaacatt gcgcactgca
     6481 aaaaaacatg ctgttcgaat taatagtggt tggggctcgt agaagctgct aacgtgcttg
     6541 gcataattta tagaagcagg tcaggcggaa tggcacttcc ttattccaga tgcgtgcgga
     6601 ttatgcatga ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgcttgtgg
     6661 tggatcacag ttattggaga cttccttggc cgtacgatgg gttttgtttc aggtatctgg
     6721 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     6781 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     6841 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     6901 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     6961 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     7021 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     7081 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     7141 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     7201 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     7261 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     7321 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     7381 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     7441 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     7501 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     7561 ctctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     7621 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     7681 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     7741 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttg aattgccatt
     7801 atcaacaagt ttgtacaaaa aagcaggctc cgaattggta tcacgaggac gatttgaagc
     7861 cccctaccca tttacgctga cttgacggga cggcggcttt gttgaataga gcgtcagacc
     7921 ccgtagaaaa gatcaaagga tcttc
//
