LOCUS       sequence-472873-        7432 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty backbone to generate guideRNA shuttles, that also drives 
            ubiquitous expression of nuclear mKate..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7432)
  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 7432)
  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-Spe-KpnI-ubb-nmKate
FEATURES             Location/Qualifiers
     source          1..7432
                     /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             4748..4768
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4769..5467
                     /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"
     polyA_signal    5472..5606
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5496..5515)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5550..5569
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5830..5847)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5830..5846)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5839..5861)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6055..6074)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6174..6196
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6234..6252)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6320..6424
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6425..6493,6494..7285)
                     /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(6643..6662)
                     /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 ctggatcact agtaaagccc atcgtctcga gggtccttag actggtacca ccagcaaagt
     1261 tctagaattt gtcgaaacat ttatgttata tatttcctga aaaaaattct gagtaagttc
     1321 ttaagtgtta ttgccagcaa cataaacaac agacggcaaa atgaataaat gataacaaag
     1381 cagtaggctt aaataaacct aatttttata ggctgttctc tacaaccctc aaacagtgat
     1441 tagttttgta cttataaact tgccctttca ttcatatttc aagaaaattg gttcagaaga
     1501 tctggatatt ctagcagttg ttcaagctca tggagggatc agtgacctga ttccacaatg
     1561 actaggccta atccagaaat tagatgactg tcaacataaa aaggcacagc actcactagc
     1621 tgccctatat attttattat attttacata tattatttta tttatttagc tctgagtgct
     1681 gtactttctg gttaaagaaa actgcttaca acagctaacc tgtactacct caggctcagg
     1741 gaatttggaa caggtttgtc tggtttgttt ctttaaccat gcatgcttgt tttcaactat
     1801 ggcaacacag tcacatggga cattacagaa atgatttgtc gatgacatgc gacttttctt
     1861 taataaagcg caaagatccc aaaaagcaaa cttttaacaa aaatcatata attatatttt
     1921 caatccagct ttgtagcaac tttgtgctgc tgttcactca gcaacagata gtcagtataa
     1981 ggtcagtgtg tctcaaagca gtgccatctg tttcacacat tgcgttctat atataagtgt
     2041 gctggttgac acgacactgt ataaggccta ggctaaaaca caaacaatgt agaatgacac
     2101 tgtgtttttt ttgtaaacaa atgttgtttt tggttaaaca tctttgtgaa aacatcctcc
     2161 tgtcatgtat ttgctatatt caaatgttaa acccgtgcag aatagaacat atacaaaaaa
     2221 aaacaacaca acacattttt aaacattatt aaatatcaag tattgctggc agttctgttt
     2281 ctgttttaca gtaccctttg ccacagttct ccgcttttcc tggtccagat tccacaagtc
     2341 tgattcacaa atagcaaagc gaataaacaa ccaaagcagc caatcactgc ttgtagactg
     2401 tcctgcgaga ccggcccatt ccagaacatt ctggaaactt cctttatatg ataattataa
     2461 atacatttaa attattgata caaaacatgt aattcctaga acataaccat agcaatcatt
     2521 agttttcagg gtaattatgt atttttagga tttgactgcg gaaagatctg gtcatgtgac
     2581 gtctcatgaa cgtcacggcc ctgggtttct ataaatacag taggactctc gaccatcggc
     2641 agatttttcg aagaagaaga tcagtttcag gagccgtact gttccgtttc aacgcaaata
     2701 ttaacggtaa gagcgaattt cctagtttgt tttcatgcca ttctttaaaa ccatagcgta
     2761 ttactttaat tatagtaaac tttcgctttc tttattacaa gagacgtttt gtgttgattc
     2821 tccgcggaca ttttcggtca gacaatcaga aaatgaccgc ggaggaccag taacttgcat
     2881 tacacgtaag ttaaatcttc gtgtattaaa atggttaggt tgttaacgtc aaataggtta
     2941 ccgtgtttgc gtgtgatcag gttggttttg ttagattttt gtcagtattt ttaatttatt
     3001 tgttttagtt tatttatttt ttttgctgaa tcatagtttg tgaacaaaga acccggatgt
     3061 tacatacagt acagccgcca tgttacagag agttataact taatcatttt aaaaataatt
     3121 ttgccttact tttagtttgt catgttgaga aatgaggaaa tgttaaaatg aggaaatatc
     3181 caattaattt aatatatcaa aataatccat gattacaatg cactgaactg gagaaaatta
     3241 agatgttttc tagtgtcatg aaacaaatgt aagagatgta cattgtagat gttttatgtc
     3301 aagaattggc tagttgatgc agcatactgg cgatactcag ttgtaataac agtaacgtta
     3361 catgttaata gactactgag tatgctgttc tgtctatgta tgctctgtaa gctacgagaa
     3421 ggactttttt aaacagtaaa gggtgcaata tttttacaaa ttgaattaaa taaaggctgt
     3481 ctattaagta atatgcttga tatttttctt acttgatcgg aaataagaaa aaatataaac
     3541 gttgttgctc taaaaatcct agttcagttt agccaaccac aaataccttt ttgttcctcc
     3601 aacagttttt ttttcttctc tataatattt ggcagtctat agtactccaa atgtttcccc
     3661 acagtctaac taattggtac agccaaaatc atgacactta ttgcaataat aattttggtt
     3721 cattggcatt gttgatagcc tgtgccacta atatggtcga ttgatcatgc ttcaggaaga
     3781 aaactatatt gtttgatgta agattattaa atcttcacct gcttccatta caaactattc
     3841 ccatcttatt gaattctggt atgtcttaaa ggattagttc acttcccaaa tcaaaattta
     3901 cttagccttt tttcatccat gatccctttt tttcatcatt aatgaagaaa ttgtttttga
     3961 aaaagtttca agattttttt ctctatattg tggagcttgt tagtttaaaa ttccaaaatg
     4021 caatatgtgg cttcaaatgg ttctaaatga tcccagtcaa ggaataacag tcttatctaa
     4081 tgaaaccatt agacctttaa aaataaaaaa aataaaagta tttattttta aatgactgag
     4141 tgattaagtt gaatttcagc gtttccttac tgtgtagaag tccttcctta ctggccccac
     4201 cctttggttc tctgccaatc tgctacctaa tgtaatgttg tggaacatta ttattcttta
     4261 tttcttaatt ttttattttt tattttaaaa caatgtaaac tgcacagatg tgcagtttgt
     4321 ttaaaatggc caatgctttg gaaatgcatg acataattag atttcatgat gcacaaagcc
     4381 aaatctcaga gcttgtgcaa aatgagctat catttcacta ggtaagaccc taaattttca
     4441 tataggatca tttggacaat tttgctgcag gtaaaatgca ttctatagtc cactgtcagc
     4501 cattgttttg gatagtattt atttttctct acaagtatag tcaatagttt tctattattt
     4561 taaaggtttg taacatttaa gggtgaccaa atgcaaagta aaatttcatt ttcgggtgaa
     4621 ctatctcgtt taacatggga gaagtgcaaa acatacatta ttggctagaa cattgtagta
     4681 ttttttaaat ggaaatgtgt gattgctaat cttactttga atttgtttac agggatccgc
     4741 caccatgcca aagaagaagc gtaaggtagt gagcgagctg attaaggaga acatgcacat
     4801 gaagctgtac atggagggca ccgtgaacaa ccaccacttc aagtgcacat ccgagggcga
     4861 aggcaagccc tacgagggca cccagaccat gagaatcaag gcggtcgagg gcggccctct
     4921 ccccttcgcc ttcgacatcc tggctaccag cttcatgtac ggcagcaaaa ccttcatcaa
     4981 ccacacccag ggcatccccg acttctttaa gcagtccttc cccgagggct tcacatggga
     5041 gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca
     5101 ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcccat ccaacggccc
     5161 tgtgatgcag aagaaaacac tcggctggga ggcctccacc gagaccctgt accccgctga
     5221 cggcggcctg gaaggcagag ccgacatggc cctgaagctc gtgggcgggg gccacctgat
     5281 ctgcaacttg aagaccacat acagatccaa gaaacccgct aagaacctca agatgcccgg
     5341 cgtctactat gtggacagaa gactggaaag aatcaaggag gccgacaaag agacctacgt
     5401 cgagcagcac gaggtggctg tggccagata ctgcgacctc cctagcaaac tggggcacag
     5461 agtttaaata ggatccagac atgataagat acattgatga gtttggacaa accacaacta
     5521 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     5581 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     5641 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg
     5701 ctgattatga catttcagtt gacgaagaca aacaaagttc tgttgtgact atgggggggg
     5761 gggcgcctgg ggatggtctc gcccggggag taattcaggg tagaaccgcc actgccttta
     5821 gcgacattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     5881 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     5941 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     6001 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
     6061 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     6121 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     6181 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     6241 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     6301 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     6361 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     6421 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     6481 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     6541 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     6601 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     6661 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     6721 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     6781 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6841 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6901 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6961 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     7021 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     7081 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     7141 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     7201 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     7261 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     7321 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     7381 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc cc
//
