LOCUS       sequence-424299-        6951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA targeting to the intergradtion site E4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6951)
  AUTHORS   Jiang W, Wang S, Avila P, Jorgensen TS, Yang Z, Borodina I
  TITLE     Combinatorial iterative method for metabolic engineering of Yarrowia
            lipolytica: application for betanin biosynthesis.
  JOURNAL   Metab Eng. 2024 Sep 9:S1096-7176(24)00110-1. doi: 
            10.1016/j.ymben.2024.09.003.
  PUBMED    39260817
REFERENCE   2  (bases 1 to 6951)
  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: "Metab Eng. 
            2024 Sep 9:S1096-7176(24)00110-1. doi: 10.1016/j.ymben.2024.09.003."
COMMENT     Sequence Label: pCfB11785
FEATURES             Location/Qualifiers
     source          1..6951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(2052..2085)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(2555..2572)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2555..2571)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2670..2687)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2841..3429)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2921..2940)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3551..4342,4343..4411))
                     /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     4174..4193
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4412..4503)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4545..4564
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4570..4589
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    4796..4829
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_RNA        complement(4893..4967)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(join(6831..6951,1..908))
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(join(6831..6951,1..908))
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
ORIGIN
        1 ccggctccgg atcggacgat tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa
       61 ttgccgtcaa ccaagctctg atagagttgg tcaagaccaa tgcggagcat atacgcccgg
      121 agccgcggcg atcctgcaag ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc
      181 atacaagcca accacggcct ccagaagaag atgttggcga cctcgtattg ggaatccccg
      241 aacatcgcct cgctccagtc aatgaccgct gttatgcggc cattgtccgt caggacattg
      301 ttggagccga aatccgcgtg cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc
      361 atcagctcat cgagagcctg cgcgacggac gcactgacgg tgtcgtccat cacagtttgc
      421 cagtgataca catggggatc agcaatcgcg catatgaaat cacgccatgt agtgtattga
      481 ccgattcctt gcggtccgaa tgggccgaac ccgctcgtct ggctaagatc ggccgcagca
      541 atggcatcca ttgcctccgc gaccggctgc agaacagcgg gcagttcggt ttcaggcagg
      601 tcttgcaacg tgacaccctg tgcacggcgg gagatgcaat aggtcaggct ctcgctgaat
      661 tccccaatgt caagcacttc cggaatcggg agcgcggccg atgcaaagtg ccgataaaca
      721 taacgatctt tgtagaaacc atcggcgcag ctatttaccc gcaggacata tccacgccct
      781 cctacatcga agctgaaagc acgagattct tcgccctccg agagctgcat caggtcggag
      841 acgctgtcga acttttcgat cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt
      901 ttacccattg ctgtagatat gtcttgtgtg taagggggtt ggggtggttg tttgtgttct
      961 tgacttttgt gttagcaagg gaagacgggc aaaaaagtga gtgtggttgg gagggagaga
     1021 cgagccttat atataatgct tgtttgtgtt tgtgcaagtg gacgccgaaa cgggcaggag
     1081 ccaaactaaa caaggcagac aatgcgagct taattggatt gcctgatggg caggggttag
     1141 ggctcgatca atgggggtgc gaagtgacaa aattgggaat taggttcgca agcaaggctg
     1201 acaagacttt ggcccaaaca tttgtacgcg gtggacaaca ggagccaccc atcgtctgtc
     1261 acgggctagc cggtcgtgcg tcctgtcagg ctccacctag gctccatgcc actccataca
     1321 atcccactag tgtaccgcta ggccgctttt agctcccatc taagaccccc ccaaaacctc
     1381 cactgtacag tgcactgtac tgtgtggcga tcaagggcaa gggaaaaaag gcgcaaacat
     1441 gcatgcatgg aatgacgtag gtaaggcgtt actagactga aaagtggcac atttcggcgt
     1501 gccaaagggt cctaggtgcg tttcgcgagc tgggcgccag gccaagccgc tccaaaacgc
     1561 ctctccgact ccctccagcg gcctccatat ccccatccct ctccacagca atgttgttaa
     1621 gccttgcaaa cgaaaaaata gaaaggctaa taagcttcca atattgtggt gtacgctgca
     1681 taacgcaaca atgagcgcca aacaacacac acacacagca cacagcagca ttaaccacga
     1741 tgaacagcat gacattacag gtgggtgtgt aatcagggcc ctgattgctg gtggtgggag
     1801 cccccatcat gggctctcgt ccagccgcgg aaaccggaaa cgctcgaatc tgccgccgct
     1861 catactcacc gaaacgtgtg gggctcgaaa aaacgggcgc caaactcctt atactactgt
     1921 atattcaagc aagtatatcc gtgggtgcgg gtgatttgga tctaaggttc gtactcaaca
     1981 ctcacgagca gcttgcctat gttacatcct tttatcagac atagcggccg caaatttaaa
     2041 taaaatgaag tgaagttcct atactttcta gagaatagga acttctatag tgagtcgaat
     2101 aagggcgaca caaaatttat tctaaatgca taataaatac tgataacatc ttatagtttg
     2161 tattatattt tgtattatcg ttgacatgta taattttgat atcaaaaact gattttccct
     2221 ttattatttt cgagatttat tttcttaatt ctctttaaca aactagaaat attgtatata
     2281 caaaaaatca taaataatag atgaatagtt taattatagg tgttcatcaa tcgaaaaagc
     2341 aacgtatctt atttaaagtg cgttgctttt ttctcattta taaggttaaa taattctcat
     2401 atatcaagca aagtgacagg cgcccttaaa tattctgaca aatgctcttt ccctaaactc
     2461 cccccataaa aaaacccgcc gaagcgggtt tttacgttat ttgcggatta acgattactc
     2521 gttatcagaa ccgcccaggg ggcccgagct taagactggc cgtcgtttta caacacagaa
     2581 agagtttgta gaaacgcaaa aaggccatcc gtcaggggcc ttctgcttag tttgatgcct
     2641 ggcagttccc tactctcgcc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2701 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2761 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2821 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2881 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2941 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3001 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     3061 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3121 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3181 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3241 gagttcttga agtggtgggc taactacggc tacactagaa gaacagtatt tggtatctgc
     3301 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3361 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3421 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgacgcg
     3481 cgcgtaactc acgttaaggg attttggtca tgagcttgcg ccgtcccgtc aagtcagcgt
     3541 aatgctctgc ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3601 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3661 catctggccc cagcgctgcg atgataccgc gagaaccacg ctcaccggct ccggatttat
     3721 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3781 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3841 gtttgcgcaa cgttgttgcc atcgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3901 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3961 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4021 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4081 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4141 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4201 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4261 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4321 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4381 taagggcgac acggaaatgt tgaatactca tattcttcct ttttcaatat tattgaagca
     4441 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4501 aaataggggt cagtgttaca accaattaac caattctgaa cattatcgcg agcccattta
     4561 tacctgaata tggctcataa caccccttgt ttgcctggcg gcagtagcgc ggtggtccca
     4621 cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggact
     4681 ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     4741 ctgggccttt cgcccgggct aattatgggg tgtcgccctt attcgactct atagtgaagt
     4801 tcctattctc tagaaagtat aggaacttct gaagtgggga tttaaatgcg gccgcgaatg
     4861 cacgcgatac cgtacccaca caaaaaaagc accaccgact cggtgccact ttttcaagtt
     4921 gataacggac tagccttatt ttaacttgct atttctagct ctaaaaccgg tgtatcctgt
     4981 ccctctctgc gccgacccgg aatcgaaccg ggggcccatc gatggcaacg atggatttta
     5041 ccactaaacc atcggcgcgt ttcttggctt ttgttaggaa ccaacctcga aattactcag
     5101 cagtttcctt aggttagggc ggaaatcgag tctggataag acgtggaacg attgatacga
     5161 ttacttgatc tgttagcaat gattcactat cgcacgcatt ccgttgcatc aaatttaggg
     5221 atgccatcaa ctttcagttc ataattaata tcttaccaaa ttaggtaatc tgcaaaagtt
     5281 cagactgtga aatgtaacat tttatatatc aagctctatt taatgcctca cagtagttaa
     5341 cataaagaga tacagaattg tcgtgtcagt gtatactatc catgtgtata ctctggatat
     5401 ccatttgtat tccattatct acgaattggc ggggttctgc tcgagctggg tggcgaggtt
     5461 gttaagtcgg tccatttttg tgtgtcttgg ttggatgagg tccaaagtgg ggggagtcta
     5521 ccaggcttta tatacacctg gtagttgcaa ctttcggtat gatccccacg aaagataaag
     5581 ttcagcatgt ggagagccga ggtagtatgc gtgtgacaga gacccaaact gggggtcacc
     5641 gcgtgcacac tggcgacgcc gctctggaag gtcacatgac cgcagatact ccgttctccc
     5701 ctcacgtcga ttcggctcgg ggacacggtc agcgccattg ccacgtgtcc acggttctct
     5761 ccactctgta gaaagtcact tccaccaagt tccatgaagc ttattaggtt tttggagaaa
     5821 tccggggcgg catttctggg gtaacttggg caagccagta gtggatcaat tgggacccga
     5881 attgggtgga aaaaggtgat aacgagccct ttgggatccc cacaattgat ctaagacctc
     5941 tgcttgcccc gatcttggcc ccctcagccc caacactgtt gctcttcaaa ctcccccata
     6001 cctcggttca gatgcagcct tcaaaacttt aacgtacctc cgagcctgtg aaaacgtggt
     6061 catgaaaaaa cgagagatgt aagtcccgtc acataccgct gcgagaccac cagctgtaca
     6121 tacacttggc gttccagcat gcacggtcgc tcaacaggcg ctcccccatt ggcggaaaga
     6181 caagccccca ctagattcct gttcaagggc aaagtctaga agcccgcccc ccccagcgcc
     6241 cgtttccttg ttagtgcggc ctgcacatgt cacctgtctg gcaggtatcg tcgctacttt
     6301 gccagtcgta tcgtcgtcaa cggattgcgc tgcccattca cgtgtaaact ctcgacggat
     6361 atcccggagt ttcccgccga tcctcccgcg agccactgcc acgtcgcctc caagcctgct
     6421 gaattcggac acgggcatct cacttgcgta tgtatggaaa ataaaaatac gacagtcgaa
     6481 cataaaggga tgacgagtag tcacagaggg aattgatgaa caaagacggg attttgccac
     6541 ctacaagcca gattttctat ttacaacaat atctggtcaa atttcagttt cgttacataa
     6601 atcgttatgt caaaggagtg tgggaggtta agagaattat caccggcaaa ctatctgtta
     6661 attgcctaaa tgccaacacc gttgtaggca acagcgttgg gagagccctt gaggttggtg
     6721 gtgggaagaa caccggtgtt ggactcagta ataagagctt cgacctgggc aggggtgaga
     6781 acctcgtcat tgatggacag gtagtaggag gcaagaccgg caacgtgggg ttattccttt
     6841 gccctcggac gagtgctggg gcgtcggttt ccactatcgg cgagtacttc tacacagcca
     6901 tcggtccaga cggccgcgct tctgcgggcg atttgtgtac gcccgacagt c
//
