LOCUS       sequence-330627-        5823 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. pyogenes Cas9 guide RNA expression in tilapia cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5823)
  AUTHORS   Hamar J, Kultz D
  TITLE     An efficient vector-based CRISPR/Cas9 system in an Oreochromis 
            mossambicus cell line using endogenous promoters.
  JOURNAL   Sci Rep. 2021 Apr 12;11(1):7854. doi: 10.1038/s41598-021-87068-3.
  PUBMED    33846462
REFERENCE   2  (bases 1 to 5823)
  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: "Sci Rep."; 
            date: "2021-04-12"; volume: "11(1):7854. doi"; pages: " 
            10.1038/s41598-021-87068-3"
COMMENT     Sequence Label: TU6m-gRNAscaffHygroR
FEATURES             Location/Qualifiers
     source          1..5823
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1099..2124
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1099..2124
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    2247..2368
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2284..2303)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2338..2357
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3010..3030)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3010..3028)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3049..3065)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3049..3065)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3062..3084)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3073..3089
                     /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(3097..3103,3104..3121,3122..3127))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3142..3163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3280..3297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3451..4039)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3531..3550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4210..5001,5002..5070))
                     /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     4833..4852
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5071..5175)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5201..5656)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5338..5359)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5550..5569
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5783..5805
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5797..5814
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5798..5814
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg cgaattgggt accgggcccc ccctcgaggt cgacgttgca
       61 gggtttcatt cggcggcgtg ctgacgtttt ttgcaggaat gtgtgtgatt tgcggggttt
      121 tgggccgggg agggtgacgg gaaatgaggc cgcctcccat tgagtataaa acctcccagt
      181 ccggcccctg tcttcctctt tctgttacct ggcttagggg cagcagcttt ggccgtgaaa
      241 acccagaaac accggtgagt ttagctccca ttcttcacat tgagagggac ccggcctcgg
      301 gcctttaaag gttttagtta aacgtgattt gttcaggcgt tagcatcctc tttgtgcttc
      361 tttcttttac tcccaaactt tgctttttcg gttacgtaac ttgacactaa cctgtagatt
      421 taattttcga gggaattaac cctaaaaaaa atagtaaagg tcgttctcgc ctgattgtat
      481 ttaacatgat agttttttgt gaggtttgtg cgttagtgga tttactagat gctgtttttt
      541 aaacttttga cgaaagattt tttacgaggg aggagaaaat gtagagtggg caacgggaac
      601 aaaggagggc tgcaagaccg gagatccgaa gatgcttgaa tggcatttct tctctctcct
      661 cactgaaaca actgcccgta tttatccggc aacaggtgct tgaagtccgg acaagatggc
      721 tttgaatatt tggcgatctt tgtaacttta tccgacgtgg cccagtggag taacacgtgt
      781 ctcggctggc ttgtggccga gtgaggccta gcagtagcag ccgcctggta gcggctaaag
      841 cctagcgact gggtagccat tgagttttta acttagtata aattaaaaaa aaaacaatac
      901 gttttagtag gctttctttt ttttttttaa acgcctttca ttaggcctat atgccgggat
      961 ggtttcctaa tgtagctggg ctcaagatgc gatggtttag ttgtacatgc acatctgtta
     1021 acgaagcttg acggctactt aatattaagc ctaacgcaat ttttatttac ttgcccacag
     1081 aaactaaaac catgagatat gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     1141 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     1201 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     1261 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     1321 ccggaagtgc ttgacattgg ggaatttagc gagagcctga cctattgcat ctcccgccgt
     1381 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     1441 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     1501 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     1561 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     1621 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     1681 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     1741 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     1801 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     1861 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     1921 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     1981 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     2041 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     2101 actcgtccga gggcaaagga atagagcggc cgcgactcta gatcataatc agccatacca
     2161 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     2221 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     2281 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     2341 gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta
     2401 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     2461 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt cccggtctga
     2521 agtatactat gtgccgaatt tcccaccaag tggtaatgag caatgtttgt tattatggct
     2581 cactattact tttgttctgc tgccttcagc cgtccaatca gaagcgtgtg agtaaacccg
     2641 gtctgtgtaa attacccaca atgcctccgt gtgtctcact cacaggctca aagtgaaagc
     2701 agagaatggg gaacagactc agactggcgc tctctgagcc tctgtgtctt cttacagatg
     2761 tataaccgac ccagcagcac ccagacatgt ttctgctgct gtttcccatc agtttaaacg
     2821 cagtttaagc gtcttccccc tgtaaccgtg actcacacag cttccaggac agagggcttt
     2881 aaattgctcc cgggtccttg gagctatcga tgtgtgtcta gaaatagcaa gttaaaataa
     2941 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgagctccag
     3001 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt
     3061 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     3121 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     3181 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3241 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     3301 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3361 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3421 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3481 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3541 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3601 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3661 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3721 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3781 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3841 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3901 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3961 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4021 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4081 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4141 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4201 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4261 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     4321 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     4381 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     4441 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4501 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4561 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4621 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4681 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4741 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4801 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4861 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4921 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4981 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5041 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5101 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5161 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     5221 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     5281 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     5341 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     5401 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     5461 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     5521 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     5581 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     5641 ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg
     5701 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     5761 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc
     5821 gcg
//
