LOCUS       sequence-476625-        6610 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Drives expression of 3 different gRNAs targeting tnnt2a, and 
            expression of nuclear mKate in cardiomyocytes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6610)
  AUTHORS   Keeley S, Fernandez-Lajarin M, Bergemann D, John N, Parrott L, 
            Andrea BE, Gonzalez-Rosa JM
  TITLE     Rapid and robust generation of cardiomyocyte-specific crispants in 
            zebrafish using the cardiodeleter system.
  JOURNAL   Cell Rep Methods. 2025 Mar 24;5(3):101003. doi: 
            10.1016/j.crmeth.2025.101003.
  PUBMED    40132543
REFERENCE   2  (bases 1 to 6610)
  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"; journalName: "Cell Rep 
            Methods."; date: "2025-03-24"; volume: "5(3):101003. doi"; pages: " 
            10.1016/j.crmeth.2025.101003"
COMMENT     Sequence Label: pTol1-U6abc_tnnt2a_cmlc2-nmKate
FEATURES             Location/Qualifiers
     source          1..6610
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      24..612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     513..532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     766..783
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    900..921
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(936..941,942..959,960..966)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    974..990
                     /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     979..1001
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     998..1014
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     998..1014
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3693..3713
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3714..4412
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHRV"
     regulatory      4414..4423
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(5008..5025)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5008..5024)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5017..5039)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5233..5252)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5352..5374
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5412..5430)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5498..5602
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5603..5671,5672..6463)
                     /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(5821..5840)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
       61 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
      121 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
      181 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      241 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      301 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      361 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      421 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      481 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      541 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      601 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      661 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
      721 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
      781 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
      841 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
      901 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
      961 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     1021 tacgccaagc ttgcatggtc cctttagcca gtagcggttc taggcacggg ccgtccgggc
     1081 ggtggcctgg ggcggaaaac tgaagggggg cggcaccggc ggctcagccc ttgtaatata
     1141 ttatatgcac cactattggt ttacttatgt cacagtttgt aagtttgtaa cagcctgaac
     1201 ctggatcagc ttaggccttc agtaagtctc tcagcgtttc tccagcctcg gtcattcagt
     1261 aagacgagtt tcctgctctc ggcgtctttt gttctggtct tcaaggaggg gggaaatgtt
     1321 ctgcgcatgc ctgtggggga gggagaagga cacgtcactg aaaacgtccc tgcatcacac
     1381 cgagacaccc aatcactcaa gccaagacca gataattttg catatgcttt acagtttgaa
     1441 aattaccacg gtaatccctc acacaaactc tggatttgag atccttcagg ttttatcagt
     1501 ttgcaggttt atgtcaccat gatatagggt cagacttgat ctaagggagc tgaataagtg
     1561 gtttagtcac tcaccacctc ccaaaaacat acccagaagt ccctggtata tatagctctc
     1621 cctccagctc ttggttcggg cttagaagag caagtgggtt taagagctat gctggaaaca
     1681 gcatagcaag tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     1741 tgctttttta gtcatctcat taccctccac gtgtctgtct gggttttcat gggctctgct
     1801 ctagtgagag cagcttcctt ttgtcggagt gttttgtgct cttatccaag gcagggggga
     1861 tctgcgcatg cctgtggggg gaggagaagg acacgtgaac aaaagctcct cgatgtcaca
     1921 caggaagttc aggaactcat ccaatcactc taaagaaacg gcctgtttcc ttcgcatacg
     1981 cttacagctc caaaactcta cggtaaacct acataaactg ctggttttca aattttaaag
     2041 aatttaaggg tttacaggtt tactactaca cagtgattta ctgacacatg taagtgtaaa
     2101 tgagttgaat aagtaagtaa gccatatacc acacatgaaa cacataccca gaagtcactg
     2161 gtatatatag ccgtcctcca gactcccagt tcgggagatg aagagaccac tcgtttaaga
     2221 gctatgctgg aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa
     2281 gtggcaccga gtcggtgctt tttttaaacc aaatccaatc cgagagtctg tgaatgtttc
     2341 gatgggccgt ttgctggcgt ctgagcatgt tgtcctctgg tctgcggcgg gggaaggtcc
     2401 gcgcatgcct gtgggggaag gcgaaggaca cgaggacaaa agatcagtta cgtcacagaa
     2461 gaactcatcc aatcacacaa gcctaaaacc agcttcctct gcatatgctt tacagttcaa
     2521 aaatactacg gtaaacctct acaaaactgc tggtttcaaa ctttgaaggt tttaagcgtt
     2581 tgcaggtttg ccccgaagag gtttactgtc atgtttgagg taaatgagtt gaataagtag
     2641 gtttatccac ttaccacatg gcagaaacat acccagaagt cccgggttta tatagcagtt
     2701 ctccaggctc ctagttcgga ggaaagagct cgcaagggtt taagagctat gctggaaaca
     2761 gcatagcaag tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     2821 tgcttttttt aaacctgact ccaaatcagc agacttaaca ttctttaaaa tgattgattc
     2881 aatagtgata aaaatcaggc atagccagtt gtaactttag ataaattaca gaaaatgtca
     2941 aatacagaga accgattctt ttttatgata catccaagca cacatttaac acaatccagg
     3001 caaaccccga atttcacagt cacaagcact gtttgtacaa gagctttgcc taaggacaca
     3061 cagtctctat aagtccaggt cgttggtttc actcttattt taaacatgtg acatttttcc
     3121 tgccatcctg tcttaggctg ctgtttgctt cattccatgt cacattaatt tcctcagtag
     3181 caccttttac acacacagcc aatcttttcc agaaaattca attgctttga aaagataatg
     3241 tgtgaacaaa tccatttaga aaaggaaaat taagaatttg taaaatcatc tgtaaattgt
     3301 tggcattctt ctgtatatga acatcacatc atttacaggt aaaggtctgg tcattaatta
     3361 tatgacaatt tactggtatt attttgtgaa aggggctatt ttcaatgcat tcatccatcc
     3421 ttttcatccc ccaaatctct cattcatgtc cccctcccca tctgcacact ttatctcatt
     3481 ttccaccctg ctggaatctg agcacttgtg cagttatcag ggctcctgta tttaggaggc
     3541 tctgggtgtc catgtagggg acgaacagaa acactgcaga cctttataga agaacaaatg
     3601 ataagagtcc tcatacataa agactccatt agaaacgtca gtgacccagg agcccagacc
     3661 aacagcaaag cagacagtga accgccacca tgccaaagaa gaagcgtaag gtagtgagcg
     3721 agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg aacaaccacc
     3781 acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa
     3841 tcaaggcggt cgagggcggc cctctcccct tcgccttcga catcctggct accagcttca
     3901 tgtacggcag caaaaccttc atcaaccaca cccagggcat ccccgacttc tttaagcagt
     3961 ccttccccga gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga
     4021 ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag
     4081 gggtgaactt cccatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct
     4141 ccaccgagac cctgtacccc gctgacggcg gcctggaagg cagagccgac atggccctga
     4201 agctcgtggg cgggggccac ctgatctgca acttgaagac cacatacaga tccaagaaac
     4261 ccgctaagaa cctcaagatg cccggcgtct actatgtgga cagaagactg gaaagaatca
     4321 aggaggccga caaagagacc tacgtcgagc agcacgaggt ggctgtggcc agatactgcg
     4381 acctccctag caaactgggg cacagagttt aaagccacca tggagatctc gagactagta
     4441 attaagtctc agccaccgtt aactgaacat gtcaaaacct gtggagactg ttgagatttg
     4501 atgttctgaa aagataaagc ctataaataa aatgttgccc aaatttcctg cctgatgttt
     4561 ttctttgtct ttgctacatg gctttgctgc tcggatcggc tcactctgtg tatgccacgt
     4621 tcactttgta ctctccttct cacggtaggt ttattatttt tagatgtgca gttagtttct
     4681 gtgaaataac acaccacaca ctgatattgt ctgtgcattg acttggtgag tgcacattgt
     4741 ttttgatctt gacatattta tatttgattg atcaggtgaa ctgtgtgaat ctaaagtgct
     4801 ccatacagat gttctgcatt gaaaatattc tcattttatt agtggaagtg agtgtatgcc
     4861 agcggccgcg gtaccctgca gtctagacat ttcagttgac gaagacaaac aaagttctgt
     4921 tgtgactatg gggggggggg gcgcctgggg atggtctcgc ccggggagta attcagggta
     4981 gaaccgccac tgcctttagc gacattcact ggccgtcgtt ttacaacgtc gtgactggga
     5041 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     5101 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     5161 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     5221 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     5281 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5341 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5401 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     5461 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5521 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5581 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5641 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5701 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5761 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5821 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5881 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5941 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     6001 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6061 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6121 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6181 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6241 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6301 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6361 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6421 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6481 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6541 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6601 gtcagacccc
//
