LOCUS       sequence-384460-        7489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transgenesis backbone. *Note: this plasmid has been discontinued. 
            Please see the new, optimized version, Plasmid 213485..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7489)
  AUTHORS   Kemmler CL, Moran HR, Murray BF, Scoresby A, Klem JR, Eckert RL, 
            Lepovsky E, Bertho S, Nieuwenhuize S, Burger S, D'Agati G, Betz C, 
            Puller AC, Felker A, Ditrychova K, Botschi S, Affolter M, Rohner N, 
            Lovely CB, Kwan KM, Burger A, Mosimann C
  TITLE     Next-generation plasmids for transgenesis in zebrafish and beyond.
  JOURNAL   Development. 2023 Mar 28:dev.201531. doi: 10.1242/dev.201531.
  PUBMED    36975217
REFERENCE   2  (bases 1 to 7489)
  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: 
            "Development. 2023 Mar 28:dev.201531. doi: 10.1242/dev.201531."
COMMENT     Sequence Label: pCK053 pDEST exorh:mCherry-
FEATURES             Location/Qualifiers
     source          1..7489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    37..160
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(201..506)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(303..322)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(826..1506)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     1421..1440
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1560..1566,1567..1584,1585..1590))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(1615..1738)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1746..1762)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(1768..2478)
                     /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"
     primer_bind     complement(1848..1871)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2046..2065)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2315..2333
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    3386..3520
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3410..3429)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3464..3483
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(3959..4146,4147..4158)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4192..4208)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4192..4208)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4245..4265)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4245..4263)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4284..4300)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4297..4319)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4308..4324
                     /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(4332..4338,4339..4356,4357..4362))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4377..4398
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4515..4532)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4686..5274)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4766..4785)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5445..6236,6237..6305))
                     /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     6068..6087
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6306..6410)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6573..6594)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6943..6954,6955..7117)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca agctatcaac tttgtataga aaagttgaac
       61 gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga
      121 ctacataata ctgtaaaaca caacatatcc agtcactatg gtcgacctgc agactggctg
      181 tgtataaggg agcctgacat ttatattccc cagaacatca ggttaatggc gtttttgatg
      241 tcattttcgc ggtggctgag atcagccact tcttccccga taacggagac cggcacactg
      301 gccatatcgg tggtcatcat gcgccagctt tcatccccga tatgcaccac cgggtaaagt
      361 tcacggggga ctttatctga cagcagacgt gcactggcca gggggatcac catccgtcgc
      421 ccgggcgtgt caataatatc actctgtaca tccacaaaca gacgataacg gctctctctt
      481 ttataggtgt aaaccttaaa ctgcatttca ccagcccctg ttctcgtcgg caaaggagcc
      541 gttcatttca ataaaccggg cgacctcagc catcccttcc tgattttccg ctttccagcg
      601 ttcggcacgc agacgacggg cttcattctg catggttgtg cttaccgaac cggagatatt
      661 gacatcatat atgccttgag caactgatag ctgtcgctgt caactgtcac tgtaatacgc
      721 tgcttcatag catacctctt tttgacatac ttcgggtata catatcagta tatattctta
      781 taccgcaaaa atcagcgcgc aaatacgcat actgttatct ggcttttagt aagccggatc
      841 ctctagatta cgccccgcct gccactcatc gcagtactgt tgtaattcat taagcattct
      901 gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac
      961 cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat
     1021 attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa
     1081 catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc
     1141 ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga
     1201 aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac
     1261 cagctcaccg tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag
     1321 aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc
     1381 cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc
     1441 aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt
     1501 ctccatttta gcttccttag ctcctgaaaa tctcgacgga tcctaactca aaatccacac
     1561 attatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgcgg ccgccatagt
     1621 gactggatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt
     1681 aatatattga tatttatatc attttacgtt tctcgttcaa ctttattata catagttgat
     1741 aattcactgg ccgtcgtttt acggtaccta cttgtacagc tcgtccatgc cgccggtgga
     1801 gtggcggccc tcggcgcgct cgtactgttc cacgatggtg tagtcctcgt tgtgggaggt
     1861 gatgtccagc ttgatgttga cgttgtaggc gccgggcagc tgcacgggct tcttggcctt
     1921 gtaggtggtc ttgacctcgg cgtcgtagtg gccgccgtcc ttcagcttca gcctctgctt
     1981 gatctcgccc ttcagggcgc cgtcctcggg gtacatccgc tcggaggagg cctcccagcc
     2041 catggtcttc ttctgcatta cggggccgtc ggaggggaag ttggtgccgc gcagcttcac
     2101 cttgtagatg aactcgccgt cctgcaggga ggagtcctgg gtcacggtca ccacgccgcc
     2161 gtcctcgaag ttcatcacgc gctcccactt gaagccctcg gggaaggaca gcttcaagta
     2221 gtcggggatg tcggcggggt gcttcacgta ggccttggag ccgtacatga actgagggga
     2281 caggatgtcc caggcgaagg gcagggggcc gcccttggtc accttcagct tggcggtctg
     2341 ggtgccctcg taggggcggc cctcgccctc gccctcgatc tcgaactcgt ggccgttcac
     2401 ggagccctcc atgtgcacct tgaagcgcat gaactccttg atgatggcca tgttgtcctc
     2461 ctcgcccttg ctcaccatga tggagaagtg gacgatcggc acacctccgc agatgaagcc
     2521 cggagagccg gccctgcgct cttatgtgaa ccccatgagt ccagattagg cagattacag
     2581 gagtgagtgg tcagattggg gtcagacagg gggataatcc agagatggag acacagcagc
     2641 gctcgctctc accacacaca ctcacgacac acacacacac actactgctg ctcatcagcg
     2701 cgtgcaggct gcgtctccat ccaaacactc ctgcacagag ctgaaatctc tcatccacag
     2761 acagacaggt gtcctgcaga acacaaccct cctgctggac aacacacaca cacacagatt
     2821 atcagactga aatattaaat ctttattaaa aacaccaaca tgagggatgt gtggatgcgc
     2881 aggagtgtgt tgagctgtga gtgtgtgtgt gtgtgtgtgt gtgtgtccag catcgctgca
     2941 atctgctttg tgttgatctg tgtaatccta tcagagctgt gctgagctgg catttactga
     3001 cggctttaca gcggctcagg gtgaatcaca ccgcggtaaa catgagtgtg tgttagcagg
     3061 actgatgatc acaccgcggt aaacatgagt gtgtgttagc aggactcatc gatgggtctg
     3121 cggcagaacg ggcagcagct gtactgaaga accaacagct catagtcctc actgtggaac
     3181 atctgagaaa cacacacaca cacacacaca cacaaccatc agacacacaa caaagcatat
     3241 agagtcaaac agacaaataa cagaggagac acacacacac acacacacag atgagcgcac
     3301 ctggaagcag gatgggcaca tggtgatgct gacgtcgggc aggagtgagc ggtagtactg
     3361 ccagctgagc ggtaccatcg atgatgatcc agacatgata agatacattg atgagtttgg
     3421 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     3481 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     3541 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
     3601 caaatgtggt atggctgatt atgatcctct agatcagatc tgcgaagata cggccacggg
     3661 tgctcttgat cctgtggctg attttggact gtgctgctcg cagctgctga tgaatcacat
     3721 acttcctcca ttttcttcca ctgattgact gttataattt ccctaatttc caggtcaagg
     3781 tgctgtgcat tgtggtaata gatgtgacat gacgtcactt ccaaaggacc aatgaacatg
     3841 tctgaccaat ttcatataat gtgaaaacga ttttcatagg cagaataaat aacatttaaa
     3901 ttaaactggg catcagcgca attcaattgg tttggtaata gcaagggaaa atagaatgaa
     3961 gtgatctcca aaaaataagt actttttgac tgtaaataaa attgtaagga gtaaaaagta
     4021 cttttttttc taaaaaaatg taattaagta aaagtaaaag tattgatttt taattgtact
     4081 caagtaaagt aaaaatcccc aaaaataata cttaagtaca gtaatcaagt aaaattactc
     4141 aagtacttta cacctctggt tcttgacccc ctaccttcag caagcccagc agatccacta
     4201 gttctagagc ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta
     4261 attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4321 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4381 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4441 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4501 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4561 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4621 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4681 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4741 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4801 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4861 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4921 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4981 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5041 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5101 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5161 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5221 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5281 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaaggcatt
     5341 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5401 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5461 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5521 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5581 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5641 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5701 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5761 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5821 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5881 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5941 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6001 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6061 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6121 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6181 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6241 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6301 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6361 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6421 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     6481 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     6541 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     6601 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccacgct
     6661 ggctaagaac tcatcagcct ccccggtcca tctacccacg taccaatgca ccaattggcc
     6721 acaatgacgg ctactacatg gtgccattcc ttcctcttta taggaatgga gactacctcc
     6781 tgtccaacaa tgctcttgga tacgagtacg cctacctgtt ggacccaggt cattgcacaa
     6841 caccagaaat gccctctgat ctgcaaaaga cgtgaatatc tgttcagaca cccatatcca
     6901 ctctgttcca cacaggtcag aggtttgtcc aggagttctt gacagaggtg taaaaagtac
     6961 tcaaaaattt tactcaagtg aaagtacaag tacttaggga aaattttact caattaaaag
     7021 taaaagtatc tggctagaat cttacttgag taaaagtaaa aaagtactcc attaaaattg
     7081 tacttgagta ttaaggaagt aaaagtaaaa gcaagaaaga aaactagaga ttcttgttta
     7141 agcttttaat ctcaaaaaac attaaatgaa atgcatacaa ggttttatcc tgctttagaa
     7201 ctgtttgtat ttaattatca aactataaga cagacaatct aatgccagta cacgctactc
     7261 aaagttgtaa aacctcagat ttaacttcag tagaagctga ttctcaaaat tgttagtgtc
     7321 aagcctagct cttttggggc tgaaaagcaa tcctgcagtg ctgaaaagcc tctcacaggc
     7381 agccgatgcg ggaagaggtg tattagtctt gatagagagg ctgcaaatag caggaaacgt
     7441 gagcagagac tccctggtgt ctgaaacaca ggccagatgg gccctcgag
//
