LOCUS       sequence-414326-        7996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ubiquitously expresses gtACR2 in zebrafish via tol2 transgenesis or 
            in vitro transcription of mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7996)
  AUTHORS   Jia BZ, Qi Y, Wong-Campos JD, Megason SG, Cohen AE
  TITLE     A bioelectrical phase transition patterns the first vertebrate 
            heartbeats.
  JOURNAL   Nature. 2023 Oct;622(7981):149-155. doi: 10.1038/s41586-023-06561-z.
            Epub 2023 Sep 27.
  PUBMED    37758945
REFERENCE   2  (bases 1 to 7996)
  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: 
            "10.1038/s41586-023-06561-z"; journalName: "Nature"; date: 
            "2023-10"; volume: "622"; issue: "7981"; pages: "149-155"
COMMENT     Sequence Label: pBJ035_MTB_gtACR2_KGC_eGFP_ER2
FEATURES             Location/Qualifiers
     source          1..7996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(25..42)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(196..784)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(276..295)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(955..1746,1747..1815))
                     /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     1578..1597
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1816..1920)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2083..2104)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2295..2314
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2458..2480
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2472..2489
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2473..2489
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    join(2548..2559,2560..2697)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    2789..2923
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2826..2845)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2880..2899
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(2973..3683,3684..3686))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     regulatory      4664..4673
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4704..4723)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4705..4723)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4731..4749)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4732..4749)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    join(7465..7652,7653..7664)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(7698..7714)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7698..7714)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(7751..7771)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(7751..7769)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7790..7806)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7790..7806)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7803..7825)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7814..7830
                     /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        complement(join(7838..7844,7845..7862,7863..7868))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7883..7904
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
       61 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
      121 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      181 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      241 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      301 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      361 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      421 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      481 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      541 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      601 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
      661 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
      721 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
      781 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
      841 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
      901 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
      961 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     1021 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     1081 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     1141 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     1201 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     1261 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     1321 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     1381 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     1441 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     1501 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     1561 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     1621 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     1681 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     1741 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     1801 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     1861 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     1921 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
     1981 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     2041 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     2101 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     2161 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     2221 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     2281 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgcg tcccattcgc
     2341 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgac
     2401 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     2461 agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgacgtc ctatagggcg
     2521 aattgggtac cggcatatgg ttcttgacag aggtgtaaaa agtactcaaa aattttactc
     2581 aagtgaaagt acaagtactt agggaaaatt ttactcaatt aaaagtaaaa gtatctggct
     2641 agaatcttac ttgagtaaaa gtaaaaaagt actccattaa aattgtactt gagtattaga
     2701 tctgcatgcg tttaaacccg ggatatcaaa aaacctccca cacctccccc tgaacctgaa
     2761 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     2821 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     2881 tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaccggtt ccggatacgt
     2941 atgaattctc acacctcgtt ctcgtaacag aacttgtaca gctcgtccat tcccagtgtg
     3001 attccagcag ctgtcacgaa ctccagcagc accatgtggt ctctcttctc gttagggtcc
     3061 ttgctcagag cgctctgtgt gctcaggtag tggttgtcag gcagcagcac aggtccgtct
     3121 ccgataggtg tgttctgctg gtagtggtca gccagctgca cgcttccgtc ctcgatgttg
     3181 tgtctgatct tgaagttcac cttgattccg ttcttctgct tgtcagccat gatgtacacg
     3241 ttgtggctgt tgtagttgta ctccagcttg tgtcccagga tgtttccgtc ctccttgaag
     3301 tcgattccct tcagctcgat tctgttcacc agtgtgtctc cctcgaactt cacctcagct
     3361 cttgtcttgt agtttccgtc gtccttgaag aagattgttc tctcctgcac gtatccctca
     3421 ggcatagcgc tcttgaagaa gtcgtgctgc ttcatgtggt cagggtatct gctgaaacac
     3481 tgcactccgt atgtcagtgt tgtcaccagt gtaggccaag gcacaggcag ctttcctgtt
     3541 gtacagatga acttcagtgt cagctttccg tatgtagcgt ctccctctcc ctctccgctc
     3601 acgctgaact tgtgtccgtt cacgtctccg tccagctcca ccaggatagg caccactcct
     3661 gtgaacagct cctctccctt gctcaccacg ttgatgtcga tctggtccag agggatgtac
     3721 tctccctcgc ttgtgattct gctcttagca gcagccttgc tcacagccac cacaggtctg
     3781 tcagcagcag atctcctgcc gaacattctg atgttgacct cgtcgttggg gggggcattc
     3841 ttctcgtggt tcagtccgaa ggggtcgtcg gcctcaggct tctcctggcc agggaggcaa
     3901 gccgggtccc actttccctc gagcactccc caggcggtgg accacaggat cacgccgtag
     3961 gtgttcttgc acaggccgtc ggcgagcaga tacaggatgg cgctcacgtt ctcgtggatc
     4021 acgcacacgc cggtggggga aatcagccac acgatgggga acatggtcca ggcgatgaag
     4081 aagatcagcc tcagcatctt gatcctcacc accaccttct gggccagagg ggtgcccacg
     4141 gactcgaagc cgtacagtcc cacctggaag atggtgaaca ccacgctgta ttcgaagacc
     4201 aggcaggtag cgccgaagaa gaagaacagc cacttcatgt actcggcggg ggagatggta
     4261 gcggtaatgc ccatcaccac cctgataatg ctggcggcct gggcgatggg gttcagaggg
     4321 atgctcttgt acttcaccag agccatgttg gagatctggc ccagcataat ggggcatgtg
     4381 cacagccagc tggccatcct ggaccaggga aagaacctgc cgtccctcat ctgcagtgtg
     4441 ccattgccgg tgctggccag ggcgtatgtg atggactcca cgaagggcag gtacacgctc
     4501 tcccagggca ccttgggggc cctgctgatc atgaagatgg tctggcatcc gctcaccacg
     4561 gcccacacca gctgcagcag gctcacgaag gtgctgtcga tcctgctcat gttgtagcac
     4621 tctgtgtggg tgctggccca ctcgccgtac acgacctggg aggccatggt ggcgggatcc
     4681 tgcaaaaaga acaagtagct tctccctata gtgagtcgta ttagtactct tctatagtgt
     4741 cacctaaata ggcctaggat ggctgaactg taaaagaaag ggaaactgtt agaattgcac
     4801 tgctgccagg gagaggcgtt tcagcaagca tgtgaccatc tggagtcaac ttcctgtttt
     4861 gctctgcatt ctacagtctg accgctggtg ggcgctagtg acagcgcgcg cctcagatat
     4921 gccagacaaa ggaagtccct ctgcattctc tcattattac cataaaaggc aatggtttga
     4981 gccgctttgc ggctgccggc ggtgtggcac tcagccagag tcgtagggct cagtaaaaag
     5041 gaacagattt cgacattcat cacaccagga ccactcccta aattttacac aaatgaagtt
     5101 tcacatcctc ttccaccctc gcctgcgatc aattacacaa ccgcatttca acgggtgtct
     5161 gccgccgggt gttactgcgt cacagatctg cagtcagtcc acggactcct cgtgagcgga
     5221 gacttcacac taaagcattt ctggacggtc actctcagtc ggttcagttc atagtctagc
     5281 ctcaaaaatc accgagatcc tgaaaaactg atgggctctg cttcgacagt cacagcggct
     5341 gaagtgcagc cagagaactc cgcagccgtt attaaagtgg accacacctc taaaattcaa
     5401 atctttcata gcggatacaa cacttcacta acgttaacgt taatcttcag cttcattaaa
     5461 gacagagggg gctgcaggct acatttaaat agaacacata acaaaaagaa aattaaccaa
     5521 gttaaaacta aatgccaata cccgttacta ataacaagat cttcgatctt ataaagcacg
     5581 aatgagccga tgttagattg atggggttta cgttacatca gatgagagct ggttagccgg
     5641 ctagctaaag taggtcacac cctgctgcag cactgcctgc acaaagaact tcccatgccg
     5701 gctatccatc aagtgtgcaa catgctcacc agcaccctca aaactgtagt taaaaatcac
     5761 atttatggct gttaaagaga gattagccag aacacaactt tatattaaga cttaacaccc
     5821 atttcacacc ggcctattca atgttcaatg tcgtgcctac gactggtcac gttaaattac
     5881 cgacattctc acctgccaca tccactaacg ctacaaaaaa gttaagatac agccaatatt
     5941 tattaaatat aacatagatt atcaaaagaa ccaccactta taaatcaaac ctagtagata
     6001 actacaaaga gaccgttatg aactgtctaa agacttcatc aaatcatatt tatccattta
     6061 cacattttac ttaacattaa tagatagaaa ctataattaa acgcttaaaa actgtaaaac
     6121 caactcacct tgggtttggg gggttgtcga cttaagagaa tgggttacaa gcagatgata
     6181 ttccgcacta gctgcgtgtg gaggagctca aagtgaggct gaaaagctgg gcgcgctctt
     6241 ttatatgacg gcacataccc ggctctagcc ataaaaggta aactttcgga gctctgtgct
     6301 ctgattggtc cagcgtcaca cacccggctg tcgcgtgcac attgctacac ttgcttcttg
     6361 caacttgaga gtgaaagaag gggaagctta actggttaaa gaacaaggcc atggcgattt
     6421 cctataaaac tggaattttt atttgcaaat tttaacacag aatttttcgt atttttctgc
     6481 gacaacgcca agactcaatt aaacttttca aaaagtctgt gcagagttac aagtggcaga
     6541 atgcgtcata atgatggatg cctccatgat cctgtgttgt gttttcaatc taacacaaca
     6601 gcagctctgt aggtcattta cctcttgtgt tcacgaaata ccgtaataca aacaatgggg
     6661 gatctaatga gtacttttcc atgtataaaa tggacatttt ttttatccaa attaggatga
     6721 cactacttgt agtatgttgt atgagcaact agctggtctt agctcagtag taatgcactt
     6781 taagaaagtg ctttaataac tttgaatgaa tgggacctgc attccatgcc acattgcttc
     6841 actatttttg tgtgtacaag ttacacaatg atgtataaag tgagtgatag cagttacatt
     6901 tttgcaagtg ggcctacctc aggtttaatg ttcattagaa atgttatatg tcaagtcttg
     6961 atcttaaata gttaaatttt ctgttcataa tttcatgtcg tgtctatgca gacataacat
     7021 ttctttttaa agaggtttat aaggataaat cctaagaaca taattctcat taaaagtaga
     7081 aaaaaaaaaa cactgaaagt gttggtgatc agctacaata aataatattt tgcacacatt
     7141 tcagatattg ccacactgaa taacagataa agctgtgctt gagcagcagt gatcatgata
     7201 aactacatgt gactttttac tgtgtaactt gggagtaatg cacatggcct tcagtgggag
     7261 ttataaacaa aaacagctga ttgtgttaaa agtgcttgct gataccaagg acaatgaggt
     7321 gatgtgactt ttattttgtt tttggcaaat tccaagtatt ttgacaagac tttttacaat
     7381 tctgagcatt ttacctctaa aatatggaag tttctcagag taaacgttga gtataggtaa
     7441 tgactaggcc ctcgagccgg gcccaagtga tctccaaaaa ataagtactt tttgactgta
     7501 aataaaattg taaggagtaa aaagtacttt tttttctaaa aaaatgtaat taagtaaaag
     7561 taaaagtatt gatttttaat tgtactcaag taaagtaaaa atccccaaaa ataatactta
     7621 agtacagtaa tcaagtaaaa ttactcaagt actttacacc tctggttctt gaccccctac
     7681 cttcagcaag cccagcagat ccactagttc tagagcggcc gccaccgcgg tggagctcca
     7741 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     7801 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     7861 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     7921 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     7981 cggggagagg cggttt
//
