LOCUS       sequence-488654-        6754 bp    DNA     circular SYN 08-OCT-2025
DEFINITION  iATPSnFR2.S29W.A95K variant with non-responsive mRuby3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6754)
  AUTHORS   Marvin JS, Kokotos AC, Kumar M, Pulido C, Tkachuk AN, Yao JS, Brown 
            TA, Ryan TA
  TITLE     iATPSnFR2: A high-dynamic-range fluorescent sensor for monitoring 
            intracellular ATP.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 May 21;121(21):e2314604121. doi: 
            10.1073/pnas.2314604121. Epub 2024 May 15.
  PUBMED    38748581
REFERENCE   2  (bases 1 to 6754)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 8, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2024-05-21"; volume: "121(21):e2314604121. 
            doi"; pages: " 10.1073/pnas.2314604121. Epub 2024 May 15"
COMMENT     Sequence Label: pAAV.SynFLEX.(cyto).iATPSnFR2.S29W.A95K.mRuby3
FEATURES             Location/Qualifiers
     source          1..6754
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      69..524
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(156..175)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     366..387
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        550..654
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(655..723,724..1515)
                     /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(873..892)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1686..2274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2175..2194
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2511..2640
                     /label=AAV2 ITR
     polyA_signal    2802..2923
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2839..2858)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2893..2912
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    3001..3589
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3107..3118
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     3516..3536
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    3661..3694
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3765..3798
                     /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)."
     regulatory      3823..3832
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     4380..4401
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4504..4525)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(5755..5788)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(5851..5884)
                     /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(5895..5914)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          5897..6029
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     repeat_region   6622..6751
                     /label=AAV2 ITR
ORIGIN
        1 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
       61 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
      121 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
      181 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
      241 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
      301 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
      361 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
      421 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
      481 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
      541 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      601 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      661 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      721 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      781 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      841 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
      901 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
      961 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1021 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1081 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1141 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1201 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1261 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1321 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1381 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1441 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1501 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1561 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1621 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1681 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1741 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1801 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     1861 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1921 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     1981 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2041 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2101 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2161 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2221 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2281 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2341 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2401 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2461 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctgcgcgctc
     2521 gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg
     2581 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
     2641 tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct aggaagatcc
     2701 ttatcgattt taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacatctcc
     2761 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     2821 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     2881 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgctcgaagc
     2941 ggccgcccgg gtcgactcta gatgcatgct cgagcggccg cccgggtcga cggtatcgat
     3001 gcggggaggc ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga agacgcggaa
     3061 gaggccgcag agccggcagc aggccgcggg aaggaaggtc cgctggattg agggccgaag
     3121 ggacgtagca gaaggacgtc ccgcgcagaa tccaggtggc aacacaggcg agcagccaag
     3181 gaaaggacga tgatttcccc gacaacacca cggaattgtc agtgcccaac agccgagccc
     3241 ctgtccagca gcgggcaagg caggcggcga tgagttccgc cgtggcaata gggaggggga
     3301 aagcgaaagt cccggaaagg agctgacagg tggtggcaat gccccaacca gtgggggttg
     3361 cgtcagcaaa cacagtgcac accacgccac gttgcctgac aacgggccac aactcctcat
     3421 aaagagacag caaccaggat ttatacaagg aggagaaaat gaaagccata cgggaagcaa
     3481 tagcatgata caaaggcatt aaagcagcgt atccacatag cgtaaaagga gcaacatagt
     3541 taagaatacc agtcaatctt tcacaaattt tgtaatccag aggttgatta tcgataagct
     3601 tctcgagtgg atcatctgca gaattcgccc ttttcagtgt ttcgaacctc ttcgagggac
     3661 ataacttcgt ataatgtatg ctatacgaag ttatgggtcg atggtgagat ctggactaga
     3721 gggtcgatgg tgatgcttgg atcaaaatag gaagaatccc aatgataact tcgtataaag
     3781 tatcctatac gaagttatac tagtggtacc accggtgata tcgccaccat gggcagatct
     3841 atgaagacta ttcacgtgag tgtcgtaact cccgacgggc ctgtatatga ggatgacgtt
     3901 gaaatggtga gcgtcaaagc aaaatggggc gagctcggta ttctcccagg ccacattccc
     3961 ttgaaagctc ccctagagat cagtgccgca cgcctgaaga aagggggcaa aacacagtat
     4021 atcgctgtgt caggcggcaa cctcgaagtg cggcctgaca aggtgaccat ctatgcccag
     4081 gcagccgaga gggctgagga tatcgatgtc ctgcgcgcca agaaagccaa ggagagagcc
     4141 gagcgccgac tccaatcaca ggtcctggtc gggaacgtct atatcaccgc cgacaagcag
     4201 aagaacggca tcaaggcgaa cttcaagatc cgccacaacg tggaggacgg cagcatgcag
     4261 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     4321 aaccactacc tgagcaccca gtccgtgctg agcaaagacc ctaacgagaa gcgcgatcac
     4381 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     4441 aagggcggta ccggagggag catgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     4501 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgcg cggcgagggc
     4561 gagggcgatg ccaccaacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     4621 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     4681 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     4741 caggagcgca ccatcagctt caaggacgac ggcacctaca agacccgcgc cgaggtgaag
     4801 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     4861 ggcaacatcc tggggcacaa gctggagtac aactttattt gtgtggacat cgattttaag
     4921 cgcgccgagc tcgcccttaa gcgcgcaatg aataggctct cagttgccga aatgaagctg
     4981 cagtctaccg gctccggaaa tgctgtgggc caggacacgc aggagcgtat ggtgtctaag
     5041 ggcgaagagc tgatcaagga aaatatgcgt atgaaggtgg tcatggaagg ttcggtcaac
     5101 ggccaccaat tcaaatgcac aggtgaagga gaaggcaggc cgtacgaggg ggtgcaaacc
     5161 atgaggatca aagtcatcga gggaggaccc ctgccatttg cctttgacat tcttgccacg
     5221 tcgttcatgt atggcagccg tacttttatc aagtacccgg cagacattcc tgatttcttt
     5281 aaacagtcct ttcctgaggg ttttacttgg gaaagagtta cgagatacga agatggtgga
     5341 gtcgtcaccg tcacgcagga caccagcctt gaggatggcg aactcgttta caacgtcaaa
     5401 gtcagagggg taaactttcc ctccaatggt cccgtgatgc agaagaagac caagggttgg
     5461 gagcctaata cagagatgat gtatccagca gatggtggtc tgaggggata cactgatatt
     5521 gcactgaaag ttgatggtgg tggccatctg cattgcaatt tcgtaacaac ttacaggtca
     5581 aaaaagaccg tcgggaacat caagatgccc ggtgtccatg ccgttgatca ccgcctggaa
     5641 aggatagagg aaagtgacaa tgaaacgtac gtagtacaac gcgaagtcgc agttgccaag
     5701 tactccaatc ttggtggtgg tatggatgaa ctttataagt aagctagcac tagtataact
     5761 tcgtatagca tacattatac gaagttatgt gtcgatggtg atgcttggca attcgggtcg
     5821 atggtgaagc attggtcttc ctattttgat ataacttcgt ataggatact ttatacgaag
     5881 ttatggggat catcacctgt ggagagaaag gcaaagtgga tgtcagtaag accaataggt
     5941 gcctatcaga aacgcaagag tcttctctgt ctcgacaagc ccagtttcta ttggtctcct
     6001 taaacctgtc ttgtaacctt gatacttacc tgatccaacg gctgcgactt ggggcagggg
     6061 gtcctagggg tggtctggcc aggagccgcg ggggcggact gcgcggtgcc caggagcaca
     6121 gctgcgctct caggcacgac acgactcctc cgctgcccac cgcagactga ggcagcgctg
     6181 agtcgccggc gccgcagcgc agatggtcgc gcccgtgccc ccctatctcg cgcctcgcgt
     6241 ggtgcggtcc ggctgggccg gcggcggcgc ggacgcgacc aaggtggccg ggaaggggag
     6301 tttgcggggg accggcgagt gacgtcagcg cgccttcagt gctgaggcgg cggtggcgcg
     6361 cgccgccagg cgggggcgaa ggcactgtcc gcggtgctga agctggcagt gcgcacgcgc
     6421 ctcgccgcat cctgtttccc ctccccctct ctgatagggg atgcgcaatt tggggaatgg
     6481 gggttgggtg cttgtccagt gggtcggggt cggtcgtcaa tttaaatact gaaaagggcg
     6541 aattccagca cactggagct cggatcttcc tagagcatgg ctacgtagat aagtagcatg
     6601 gcgggttaat cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc
     6661 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     6721 gggcggcctc agtgagcgag cgagcgcgca gctg
//
