LOCUS       sequence-401189-        4991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Zebrafish transgenics; Plasmid encodes the sgRNA2_8xCTS-ECFP 
            reporter and could be stably integrated in the zebrafish genome 
            using Tol2 transgenesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4991)
  AUTHORS   Pelea O, Mayes S, Ferry QRV, Fulga TA, Sauka-Spengler T
  TITLE     Specific Modulation of CRISPR Transcriptional Activators through 
            RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos
  JOURNAL   bioRxiv 2023.05.08.539738
REFERENCE   2  (bases 1 to 4991)
  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: "bioRxiv 
            2023.05.08.539738"
COMMENT     Sequence Label: Tol2_8xCTS-ECFP
FEATURES             Location/Qualifiers
     source          1..4991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      353..941
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     842..861
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1095..1112
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1229..1250
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1265..1270,1271..1288,1289..1295)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1303..1319
                     /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     1308..1330
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1327..1343
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1327..1343
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1362..1382
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1364..1382
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1419..1435
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1419..1435
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(1469..1480,1481..1668)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     misc_feature    1780..1871
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     1820..1839
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     CDS             join(2278..2280,2281..2283,2284..2997)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2323..2344)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2584..2603)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2931..2952
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3023..3144
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3034..3053)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3088..3107
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(3362..3499,3500..3511)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3570..3587)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3570..3586)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3579..3601)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3728..4182
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3814..3833)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4024..4045
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4208..4312
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4313..4381,4382..4991,1..182)
                     /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(4531..4550)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
       61 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
      121 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
      181 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
      241 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
      301 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
      361 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
      421 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
      481 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
      541 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
      601 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
      661 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
      721 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
      781 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
      841 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
      901 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
      961 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     1021 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     1081 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     1141 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     1201 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     1261 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     1321 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
     1381 gggaacaaaa gctggagctc caccgcggtg gcggccgctc tagaactagt ggatctgctg
     1441 ggcttgctga aggtaggggg tcaagaacca gaggtgtaaa gtacttgagt aattttactt
     1501 gattactgta cttaagtatt atttttgggg atttttactt tacttgagta caattaaaaa
     1561 tcaatacttt tacttttact taattacatt tttttagaaa aaaaagtact ttttactcct
     1621 tacaatttta tttacagtca aaaagtactt attttttgga gatcacttgg gcccggctcg
     1681 agtaatatta aggtacggga ggtacttgga gcggccgcaa taaatatctt tattttcatt
     1741 acatctgtgt gttggttttt tgtgtgaatc gatagtacta acatacgctc tccatcaaaa
     1801 caaaacgaaa caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg caggtgccag
     1861 aacatttctc tatcgatagc tagcactagt cccaggacag tactccgact tacttaatat
     1921 tttcatagcc gaggacagta ctccgactta cgagctccta ctgaccctac tagtcccagg
     1981 acagtactcc gacttactta atattttcat agccgaggac agtactccga cttacgagct
     2041 ctctaggggc ccactagtcc caggacagta ctccgactta cttaatattt tcatagccga
     2101 ggacagtact ccgacttacg agctcctact gaccctacta gtcccaggac agtactccga
     2161 cttacttaat attttcatag ccgaggacag tactccgact tacgagctct ctagactagc
     2221 ggggggctat aaaagggggt gggggcgttc gtcctgctat ctagcgtcgc gttgaccatg
     2281 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     2341 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     2401 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     2461 gtgaccaccc tgacctgggg cgtgcagtgc ttcgcccgct accccgacca catgaagcag
     2521 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2581 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2641 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2701 ctggagtaca acgccatcag cgacaacgtc tatatcaccg ccgacaagca gaagaacggc
     2761 atcaaggcca acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2821 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2881 ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2941 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaggc
     3001 cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga
     3061 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     3121 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     3181 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc
     3241 gataaggatc gagtatgtcg accgatgccc ttgagagcca attgcattca ttttatgttt
     3301 caggttcagg gggaggtgtg ggaggttttt tgatatcccg ggtttaaacg catgcagatc
     3361 taatactcaa gtacaatttt aatggagtac ttttttactt ttactcaagt aagattctag
     3421 ccagatactt ttacttttaa ttgagtaaaa ttttccctaa gtacttgtac tttcacttga
     3481 gtaaaatttt tgagtacttt ttacacctct gtcaagaacc atatgccggt acccaattcg
     3541 ccctatagga cgtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     3601 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     3661 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     3721 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     3781 gtgaccacca cacccgccgc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3841 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3901 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3961 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4021 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4081 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4141 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     4201 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     4261 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     4321 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     4381 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     4441 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     4501 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     4561 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     4621 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     4681 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     4741 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     4801 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     4861 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     4921 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     4981 tccggctggc t
//
