LOCUS       sequence-460023-        8600 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of genetically encoded calcium chelator in zebrafish 
            hepatocytes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8600)
  AUTHORS   Pozo-Morales M, Garteizgogeascoa I, Perazzolo C, So J, Shin D, Singh
            SP
  TITLE     In vivo imaging of calcium dynamics in zebrafish hepatocytes.
  JOURNAL   Hepatology. 2023 Mar 1;77(3):789-801. doi: 10.1002/hep.32663. Epub 
            2023 Feb 17.
  PUBMED    35829917
REFERENCE   2  (bases 1 to 8600)
  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"; doi: "10.1002/hep.32663";
            journalName: "Hepatology"; date: "2023-03-1- 1"; volume: "77"; 
            issue: "3"; pages: "789-801"
COMMENT     Sequence Label: fabp10a:FLAG-SpiCee-mRFP1; cryaa:mCherry
FEATURES             Location/Qualifiers
     source          1..8600
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    290..424
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(327..346)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     381..400
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    442..489
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     polyA_signal    513..720
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     709..726
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(744..1454)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     protein_bind    complement(2020..2053)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(2072..2092)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2072..2090)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2123..2140)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2123..2139)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2132..2154)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2281..2709
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2368..2387)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2578..2599
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2762..2866
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2867..2935,2936..3727)
                     /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(3085..3104)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3898..4486
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4387..4406
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4640..4657
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4774..4795
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4810..4815,4816..4833,4834..4840)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4848..4864
                     /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)."
     primer_bind     4853..4875
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4872..4888
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4872..4888
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4934..4953
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4934..4952
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      7178..7187
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7187..7210
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7820..7829
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7826..8500
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRPS
                     TGA"
ORIGIN
        1 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
       61 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
      121 gcgcggggat ctcatgctgg agttcttcgc ccaccccccg gatctaagct ctagataagt
      181 aatgatcata atcagccata tcacatctgt agaggtttta cttgctttaa aaaacctccc
      241 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
      301 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
      361 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
      421 gatccggggg taccgcgtcg agaagttcct attccgaagt tcctattctc tagaaagtat
      481 aggaacttcg ggcccgcggt ggcggcgatc tctccccagc atgcctgcta ttgtcttccc
      541 aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga cacctactca
      601 gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg gcaccttcca
      661 gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta gaaggcacag
      721 tcgaggctga tcagcgggcc gctttacttg tacagctcgt ccatgccgcc ggtggagtgg
      781 cggccctcgg cgcgttcgta ctgttccacg atggtgtagt cctcgttgtg ggaggtgatg
      841 tccaacttga tgttgacgtt gtaggcgccg ggcagctgca cgggcttctt ggccttgtag
      901 gtggtcttga cctcagcgtc gtagtggccg ccgtccttca gcttcagcct ctgcttgatc
      961 tcgcccttca gggcgccgtc ctcggggtac atccgctcgg aggaggcctc ccagcccatg
     1021 gtcttcttct gcattacggg gccgtcggag gggaagttgg tgccgcgcag cttcaccttg
     1081 tagatgaact cgccgtcctg cagggaggag tcctgggtca cggtcaccac gccgccgtcc
     1141 tcgaagttca tcacgcgctc ccacttgaag ccctcgggga aggacagctt caagtagtcg
     1201 gggatgtcgg cggggtgctt cacgtaggcc ttggagccgt acatgaactg aggggacagg
     1261 atgtcccagg cgaagggcag ggggccaccc ttggtcacct tcagcttggc ggtctgggtg
     1321 ccctcgtagg ggcggccctc gccctcgccc tcgatctcga actcgtggcc gttcacggag
     1381 ccctccatgt gcaccttgaa gcgcatgaac tccttgatga tggccatgtt atcctcctcg
     1441 cccttgctca ccatgttgtc agacctggta actccttact gtaacttatt gcacctgatg
     1501 aacaaaagat gtgaactgaa cgcaagtagg aggctaatag aggctagtac agtacaaaga
     1561 tccccaaatg atgtgtgccg gccttagtgg aggcagaccc ctggttatat acctgtgctg
     1621 cagaaagagg ggcttacaaa tggatctctc tggaatggca ttatctcatg gtgggaacag
     1681 tgttgtcagc agaatcagcg gggtggactc tgacctgaga atgacagtct agcactgaat
     1741 ggctcagaca atactcttgt ggccaactat caccggaaaa taagtgcctg ccaaaacaaa
     1801 ccagaccaga tgaagctaca tggctgtaga acgtcagtat ttaatgtgtt gtggccactt
     1861 ggttcaccag atttaagagc taattttttt aaatatcaca gataatcaat cgcttttttt
     1921 ttttcttgac tgaaaagctt agactggaaa attctttgaa aaagtttgca ctacagaaaa
     1981 taaatacata acatcctgca ctgaatgcac actattaatg aagttcctat actttctaga
     2041 gaataggaac ttcggtaccc agcttttgtt ccctttagtg agggttaatt gcgcgctagg
     2101 gataacaggg taatacgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     2161 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     2221 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     2281 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2341 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2401 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2461 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2521 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2581 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2641 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2701 acgcgaattt taacaaaata ttaacgctta cacgtcaggt ggcacttttc ggggaaatgt
     2761 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     2821 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     2881 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     2941 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3001 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     3061 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     3121 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3181 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3241 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     3301 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3361 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     3421 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3481 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3541 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     3601 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3661 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3721 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3781 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     3841 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3901 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3961 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4021 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     4081 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4141 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4201 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4261 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4321 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4381 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4441 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4501 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4561 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4621 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     4681 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     4741 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     4801 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     4861 acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgtta gggataacag
     4921 ggtaatgcgc gcgtaatacg actcactata gggcgaattg gagctctgca gaagagcatc
     4981 tctgaacaca caacacgtcc aaccttgagg cagatgggct acagcagcag aagaccacac
     5041 cgggtgccgc tcctgtcagc taagaacagg aaactgaggc tacaattcac acagactcac
     5101 caaaactgga cgatatatta ttagcccccc ttagaatttt tcatttgata atatttttct
     5161 tctggcgaaa gcctcatttg ttttatatat tatagaataa aattagtttt taatagtttt
     5221 tatgccattt taaggtcaat attattagcc cctttaagct attttttttc gatagtctac
     5281 agaacaaacc atcggtatac aatgacttgc ctaattaccc taacctgcct agttacccta
     5341 attaacctag ttaagccttt aaatgtcact ttaagctgta tagaagtgtc ttgaagaata
     5401 tctagtctaa tattattgac tgtcatcatg gcaaagataa aataaatcag ttattaaaac
     5461 tattatgatt agaaatgtgc tgaaacaatc tgctctccga taaacagaaa ttgaacaaaa
     5521 taaacagggg ggctaataaa tttaaggggt taaataattc tgattgcaaa aaaaatgatg
     5581 tctgcaaaac tgttgcaaat aatttatttg tgttgatttt aagcaaacaa attaaattta
     5641 ataaaataca acttaatctg tttgtttaaa ttcagcccta ataaattgtt tacagccact
     5701 taacgtaaaa aaattgagta aatccaagga atcatctctg aataattttt ttcagtgtat
     5761 atatatatat atatattctt acaaaacaac tcatttactt tagtttaatt ttcaggggca
     5821 aaaaactaaa gtaatcgacg ttgcttgaat aaaaagtgta attaagggaa tgaggtaaca
     5881 tttaaccatg tgtcaatgca gtttaaatat gccagttagt ggtatatgtt taaatggtaa
     5941 gctattcaaa actttaaact aacttaacca gccttttgtt gtcagactga acagactttc
     6001 catctgcatt attagagact aatctttggc tggatgaatg attcatctgc tgatatttca
     6061 gaatagacag attgaggctg tttctaatat gattatgcaa cctgagggtg attatttgaa
     6121 gcaaactcca cagaccagca ggtcattgac cgtcgtgtgt tcaaacagag cagaaacatt
     6181 tgcaaaactg gtctgacagg agaatccagt ccagcacaac acatatgctg agcaaactga
     6241 atcaatcctg caggtcaact ctcgtgcttt aagtttatta agattatttt atttatttat
     6301 tattttattt atctatttat ttatttagtt gtttatttag caattatttg tttatttatt
     6361 tgtttattta ttcctgcaga tcatgccttg tgccttttta catttaattt aatttttaat
     6421 ttaatttcct tttacttttt tttatttttt tattttattt tattttacag tctgacaaat
     6481 actsaamtaa aaccctcaga tcatgtctta tgcatttmat ttnatttttt ttnntnnttt
     6541 attttattwt ttattttatt ttattwttta ttttatttta ttttatttta ttttacagtc
     6601 tgacatactg gaccaataaa tctctcagat catgtctcat gcattttact tttattttat
     6661 tagaattaga aagatcaaag gaacaacttt taaaatatta attctgtatc aaaatctctt
     6721 ttgatacatt taattgattt aaaaaagcag ttcacccaag aaacatttcc tcacagtcga
     6781 atggttgtaa acttttatga attactttca cagaaaaaga tttttggaag aatattggaa
     6841 aaaaagcagc cattgacttc catagtaaca acaaaaaata ctatggaagt caatggctgt
     6901 tttttcacca ttcggtatct tcattctgga gcagaatttt ttgggtgatc tgtcccttta
     6961 agtcgtcaaa tcctggtgca atattccaca tgcactgacg agtctttatt tttggtctgc
     7021 tactgctgtg tgtgtgaggg catttacctc atcttctgct ggagttgatg aacggtgggt
     7081 tgttcaaaca gcagcaggtc attgactgaa ctcctctcga tataaaagct gcagatctga
     7141 agctgacctt cactttgtgt tgatcgctag cgaattcgcc accatggact acaaagacga
     7201 tgacgacaag tccggaatcg atatgtcgat gacagacttg ctcagcgctg aggacatcaa
     7261 gaaggcgata ggagccttta ctgctgcaga ctccttcgac cacaaaaagt tcttccagat
     7321 ggtgggcctg aagaaaaaga gtgcggatga tgtgaagaag gtgttccaca ttctggacaa
     7381 agacaaagat ggcttcattg acgaggatga gctggggtcc attctgaagg gcttctcctc
     7441 agatgccaga gacttgtctg ctaaggaaac aaagacgctg atggctgctg gagacaagga
     7501 cggggatggc aagattgggg ttgaagagtt ctccactctg gtggccgaaa gcatcgatct
     7561 taagatggct gatcagctga ctgaagagca gattgctgaa tttaaggagg ctttctccct
     7621 attcgataaa gatggtgacg gcaccatcac aaccaaggaa ctggggaccg tcatgcggtc
     7681 actgggtcag aacccaacag aagccgagct gcaggatatg atcaacgaag tggatgctga
     7741 tggcaatggc accattgact tcccagagtt cttgactatg atggctagaa aaatgaaaga
     7801 cacacttaag gcggatcccg ccaccatggc ctcctccgag gacgtcatca aggagttcat
     7861 gcgcttcaag gtgcgcatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     7921 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg
     7981 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc cagtacggct ccaaggccta
     8041 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     8101 gtgggagcgc gtgatgaact tcgaggatgg cggcgtggtg accgtgaccc aggactcctc
     8161 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     8221 cggccccgta atgcagaaga agaccatggg ctgggaggcc tccaccgagc ggatgtaccc
     8281 cgaggacggc gccctgaagg gcgagatcaa gatgaggctg aagctgaagg acggcggcca
     8341 ctacgacgcc gaggtcaaga ccacctacat ggccaagaag cccgtgcagc tgcccggcgc
     8401 ctacaagacc gacatcaagc tggacatcac ctcccacaac gaggactaca ccatcgtgga
     8461 acagtacgag cgcgccgagg gccgcccctc caccggcgcc tgtacaagta agaactctgc
     8521 agataaacgc tagttaatta agtaagttta aactgagttc tactaactaa cgagtaatat
     8581 ttaaattttc aggcggccgc
//
