LOCUS       sequence-384468-        7715 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 7715)
  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 7715)
  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: pCB24 pDEST crybb1:TagBFP
FEATURES             Location/Qualifiers
     source          1..7715
                     /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"
     protein_bind    complement(2681..2714)
                     /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      2867..2876
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(4081..4268,4269..4280)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4314..4330)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4314..4330)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4367..4387)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4367..4385)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4406..4422)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4419..4441)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4430..4446
                     /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(4454..4460,4461..4478,4479..4484))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4499..4520
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4637..4654)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4808..5396)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4888..4907)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5567..6358,6359..6427))
                     /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     6190..6209
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6428..6532)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6695..6716)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7169..7180,7181..7343)
                     /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 tcttcgagca
     2041 attggtgcta tcatcatgac aaagagaaaa gaaatcagtt attagaaatg agttattaaa
     2101 actattatga ttagaaatat gtttctttct cttaaacagg atttaagggg aaaatataca
     2161 gggagcgaaa aattttgact tcatctgtat atacatataa taaataatac atcaataaat
     2221 tccatctaaa tacgctacca tatatatata tatatgtata tacacacaca cacacacacc
     2281 atctaatata tgtcatgcta cattattaac ttcagtatga aatctactct gctctggagt
     2341 gtatcagcga gcgactcctc tgctcggatt atcatcacgt gacctctgac ctgaacacag
     2401 acctctctta gccataggta tacagcctat agcatagagc tcgggctgag agggaaagtg
     2461 ccgccgtcag ggctttccat tgacagtaaa atgcttacgg ccttctgggc gatgcgatga
     2521 ccctgcgctt cggcacggct gaactctgat gacggcgtgt ttctattgtc cgccgcgcgc
     2581 cgctgtgctg cccttgccag cgtataaaag ctgcggcagt ggtgaggaag gccagagcga
     2641 gccagccttc ccagcacagc tgtagtttaa acgcgttgcc ataacttcgt ataggatact
     2701 ttatacgaag ttatctgcat gctaggttgc cgggaacggt gcattggaac gcgcattccc
     2761 cgtgttaatt aacaggtaag tgtcttcctc ctgtttcctt cccctgctat tctgctcaac
     2821 cttcctatca gaaactgcag tatctgtatt tttgctaggg gatcccgcca ccatgggctc
     2881 tgaactgatt aaggagaaca tgcacatgaa gctgtacatg gagggaactg tggataacca
     2941 tcatttcaag tgtaccagcg agggtgaggg taagccctac gagggaaccc agactatgag
     3001 aattaaagtt gtggagggag gtccgctgcc tttcgccttt gacatcctcg cgaccagctt
     3061 cttgtacggc agcaagactt ttatcaacca cactcaaggt atccctgact tttttaagca
     3121 aagttttcct gagggattca cctgggaaag agtaaccaca tacgaagatg ggggagtcct
     3181 cacagccact caagatacat cccttcaaga tggttgtttg atctataacg ttaaaatccg
     3241 cggagtgaac ttcaccagca acggacccgt tatgcaaaaa aaaactctcg gatgggaggc
     3301 cttcaccgag acgttgtatc ctgcagatgg cggcttggaa gggcgcaatg atatggccct
     3361 gaagctggtt ggcggtagtc atctgatcgc taatatcaaa accacataca gaagcaaaaa
     3421 gccggcaaag aacctgaaga tgcctggtgt gtactacgtt gattaccgac tggaaagaat
     3481 taaagaggct aataacgaga catatgtgga gcagcacgag gtagccgtag ccagatactg
     3541 cgacctgcca agcaaacttg ggcacaaact taactagagc ggcccctctc gagcctctag
     3601 aactatagtg agtcgtatta cgtagatcca gacatgataa gatacattga tgagtttgga
     3661 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     3721 gctttatttg taaccattat aagctgcaat aaacaagaga tctgcgaaga tacggccacg
     3781 ggtgctcttg atcctgtggc tgattttgga ctgtgctgct cgcagctgct gatgaatcac
     3841 atacttcctc cattttcttc cactgattga ctgttataat ttccctaatt tccaggtcaa
     3901 ggtgctgtgc attgtggtaa tagatgtgac atgacgtcac ttccaaagga ccaatgaaca
     3961 tgtctgacca atttcatata atgtgaaaac gattttcata ggcagaataa ataacattta
     4021 aattaaactg ggcatcagcg caattcaatt ggtttggtaa tagcaaggga aaatagaatg
     4081 aagtgatctc caaaaaataa gtactttttg actgtaaata aaattgtaag gagtaaaaag
     4141 tacttttttt tctaaaaaaa tgtaattaag taaaagtaaa agtattgatt tttaattgta
     4201 ctcaagtaaa gtaaaaatcc ccaaaaataa tacttaagta cagtaatcaa gtaaaattac
     4261 tcaagtactt tacacctctg gttcttgacc ccctaccttc agcaagccca gcagatccac
     4321 tagttctaga gcggccgcca ccgcggtgga gctccagctt ttgttccctt tagtgagggt
     4381 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4441 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4501 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4561 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4621 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4681 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4741 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4801 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4861 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4921 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4981 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5041 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5101 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5161 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5221 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5281 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5341 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5401 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggca
     5461 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5521 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5581 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5641 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5701 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5761 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5821 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5881 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5941 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6001 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6061 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6121 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6181 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6241 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6301 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6361 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6421 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6481 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6541 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     6601 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     6661 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     6721 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     6781 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     6841 gaaccctaaa gggagccccc gatttagagc ttgacgggga aaggctggct aagaactcat
     6901 cagcctcccc ggtccatcta cccacgtacc aatgcaccaa ttggccacaa tgacggctac
     6961 tacatggtgc cattccttcc tctttatagg aatggagact acctcctgtc caacaatgct
     7021 cttggatacg agtacgccta cctgttggac ccaggtcatt gcacaacacc agaaatgccc
     7081 tctgatctgc aaaagacgtg aatatctgtt cagacaccca tatccactct gttccacaca
     7141 ggtcagaggt ttgtccagga gttcttgaca gaggtgtaaa aagtactcaa aaattttact
     7201 caagtgaaag tacaagtact tagggaaaat tttactcaat taaaagtaaa agtatctggc
     7261 tagaatctta cttgagtaaa agtaaaaaag tactccatta aaattgtact tgagtattaa
     7321 ggaagtaaaa gtaaaagcaa gaaagaaaac tagagattct tgtttaagct tttaatctca
     7381 aaaaacatta aatgaaatgc atacaaggtt ttatcctgct ttagaactgt ttgtatttaa
     7441 ttatcaaact ataagacaga caatctaatg ccagtacacg ctactcaaag ttgtaaaacc
     7501 tcagatttaa cttcagtaga agctgattct caaaattgtt agtgtcaagc ctagctcttt
     7561 tggggctgaa aagcaatcct gcagtgctga aaagcctctc acaggcagcc gatgcgggaa
     7621 gaggtgtatt agtcttgata gagaggctgc aaatagcagg aaacgtgagc agagactccc
     7681 tggtgtctga aacacaggcc agatgggccc tcgag
//
