LOCUS       sequence-384462-        7531 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transgenesis backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7531)
  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 7531)
  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: pCB59 pDEST crybb1:mKate2
FEATURES             Location/Qualifiers
     source          1..7531
                     /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"
     polyA_signal    1778..1912
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1802..1821)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1856..1875
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2683..2716)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     regulatory      2869..2878
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2875..3573
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     misc_feature    join(3897..4084,4085..4096)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4130..4146)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4130..4146)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4183..4203)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4183..4201)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4222..4238)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4235..4257)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4246..4262
                     /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(4270..4276,4277..4294,4295..4300))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4315..4336
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4453..4470)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4624..5212)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4704..4723)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5383..6174,6175..6243))
                     /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     6006..6025
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6244..6348)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6511..6532)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6985..6996,6997..7159)
                     /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 acggtaccat cgatgatgat ccagacatga taagatacat
     1801 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     1861 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     1921 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
     1981 gtaaaacctc tacaaatgtg gtatggctga ttatgatcct ctagatcaga tctcctcgag
     2041 caattggtgc tatcatcatg acaaagagag aagaaatcag ttattagaaa tgagttatta
     2101 aaactattat gattagaaat atgtttcttt ctcttaaaca ggatttaagg ggaaaatata
     2161 cagggagcga aaaattttga cttcatctgt atatacatat aataaataat acatcaataa
     2221 attccatcta aatacgctac catatatata tatatatgta tatacacaca cacacacaca
     2281 ccatctaata tatgtcatgc tacattatta acttcagtat gaaatctact ctgctctgga
     2341 gtgtatcagc gagcgactcc tctgctcgga ttatcatcac gtgacctctg acctgaacac
     2401 agacctctct tagccatagg tatacagcct atagcataga gctcgggctg agagggaaag
     2461 tgccgccgtc agggctttcc attgacagta aaatgcttac ggccttctgg gcgatgcgat
     2521 gaccctgcgc ttcggcacgg ctgaactctg atgacggcgt gtttctattg tccgccgcgc
     2581 gccgctgtgc tgcccttgcc agcgtataaa agctgcggca gtggtgagga aggccagagc
     2641 gagccagcct tcccagcaca gctgtagttt aaacgcgttg ccataacttc gtataggata
     2701 ctttatacga agttatctgc atgctaggtt gccgggaacg gtgcattgga acgcgcattc
     2761 cccgtgttaa ttaacaggta agtgtcttcc tcctgtttcc ttcccctgct attctgctca
     2821 accttcctat cagaaactgc agtatctgta tttttgctag gggatcccgc caccatggtg
     2881 agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac cgtgaacaac
     2941 caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg
     3001 agaatcaagg cggtcgaggg cggccctctc cccttcgcct tcgacatcct ggctaccagc
     3061 ttcatgtacg gcagcaaaac cttcatcaac cacacccagg gcatccccga cttctttaag
     3121 cagtccttcc ccgagggctt cacatgggag agagtcacca catacgaaga cgggggcgtg
     3181 ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaagatc
     3241 agaggggtga acttcccatc caacggccct gtgatgcaga agaaaacact cggctgggag
     3301 gcctccaccg agaccctgta ccccgctgac ggcggcctgg aaggcagagc cgacatggcc
     3361 ctgaagctcg tgggcggggg ccacctgatc tgcaacttga agaccacata cagatccaag
     3421 aaacccgcta agaacctcaa gatgcccggc gtctactatg tggacagaag actggaaaga
     3481 atcaaggagg ccgacaaaga gacctacgtc gagcagcacg aggtggctgt ggccagatac
     3541 tgcgacctcc ctagcaaact ggggcacaga tgaagatctg cgaagatacg gccacgggtg
     3601 ctcttgatcc tgtggctgat tttggactgt gctgctcgca gctgctgatg aatcacatac
     3661 ttcctccatt ttcttccact gattgactgt tataatttcc ctaatttcca ggtcaaggtg
     3721 ctgtgcattg tggtaataga tgtgacatga cgtcacttcc aaaggaccaa tgaacatgtc
     3781 tgaccaattt catataatgt gaaaacgatt ttcataggca gaataaataa catttaaatt
     3841 aaactgggca tcagcgcaat tcaattggtt tggtaatagc aagggaaaat agaatgaagt
     3901 gatctccaaa aaataagtac tttttgactg taaataaaat tgtaaggagt aaaaagtact
     3961 tttttttcta aaaaaatgta attaagtaaa agtaaaagta ttgattttta attgtactca
     4021 agtaaagtaa aaatccccaa aaataatact taagtacagt aatcaagtaa aattactcaa
     4081 gtactttaca cctctggttc ttgaccccct accttcagca agcccagcag atccactagt
     4141 tctagagcgg ccgccaccgc ggtggagctc cagcttttgt tccctttagt gagggttaat
     4201 tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4261 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4321 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4381 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4441 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4501 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4561 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4621 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4681 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4741 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4801 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4861 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4921 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4981 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5041 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5101 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5161 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5221 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aaggcatttt
     5281 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5341 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5401 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5461 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5521 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5581 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5641 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5701 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5761 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5821 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5881 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5941 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6001 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6061 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6121 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6181 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6241 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6301 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6361 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     6421 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     6481 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     6541 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     6601 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     6661 cctaaaggga gcccccgatt tagagcttga cggggaaagg ctggctaaga actcatcagc
     6721 ctccccggtc catctaccca cgtaccaatg caccaattgg ccacaatgac ggctactaca
     6781 tggtgccatt ccttcctctt tataggaatg gagactacct cctgtccaac aatgctcttg
     6841 gatacgagta cgcctacctg ttggacccag gtcattgcac aacaccagaa atgccctctg
     6901 atctgcaaaa gacgtgaata tctgttcaga cacccatatc cactctgttc cacacaggtc
     6961 agaggtttgt ccaggagttc ttgacagagg tgtaaaaagt actcaaaaat tttactcaag
     7021 tgaaagtaca agtacttagg gaaaatttta ctcaattaaa agtaaaagta tctggctaga
     7081 atcttacttg agtaaaagta aaaaagtact ccattaaaat tgtacttgag tattaaggaa
     7141 gtaaaagtaa aagcaagaaa gaaaactaga gattcttgtt taagctttta atctcaaaaa
     7201 acattaaatg aaatgcatac aaggttttat cctgctttag aactgtttgt atttaattat
     7261 caaactataa gacagacaat ctaatgccag tacacgctac tcaaagttgt aaaacctcag
     7321 atttaacttc agtagaagct gattctcaaa attgttagtg tcaagcctag ctcttttggg
     7381 gctgaaaagc aatcctgcag tgctgaaaag cctctcacag gcagccgatg cgggaagagg
     7441 tgtattagtc ttgatagaga ggctgcaaat agcaggaaac gtgagcagag actccctggt
     7501 gtctgaaaca caggccagat gggccctcga g
//
