LOCUS       sequence-424253-        6318 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA targeting to the intergradtion site E2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6318)
  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 6318)
  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: pCfB10769
FEATURES             Location/Qualifiers
     source          1..6318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(1735..1768)
                     /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(2238..2255)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2238..2254)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2353..2370)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2524..3112)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2604..2623)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3234..4025,4026..4094))
                     /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     3857..3876
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4095..4186)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4228..4247
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4253..4272
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    4479..4512
                     /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(4576..4650)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     join(6302..6318,1..2)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 acataactaa ttacatgatc atggacatgg catagacata tacaaggctt gttcaccatc
       61 tgatgcagta ccatcgtata atgcagtatc caaaccacac aaggtaaaac ccattcttct
      121 ataggcatga atagctggag cgttaacatt agtaacttcc aaccacaaat gaccagcacc
      181 tctttctcta gcaaattcag tagccaaacc catcaaagct ctaccaacac catgacctct
      241 atgttctgga gcaacttcaa tatcttcgac ggtcaatctt ctattccaac cagaataaga
      301 aacaacaaca aaaccagcca agtcaccgtc atcaccataa gcaacaaaag ttctagaatc
      361 tggatcacca tcttcaccat catcagattc gtcatcggat tcatcatctg gaaaaacctt
      421 agtcaatggt ggatcaactg gaacttctct caaagtgaaa ccatcaccag tagcagtaac
      481 tctgaaaaca gtgtcagtag tgaaagaacc atccaaagct tcaatagctt cagcatcacc
      541 tggaacagaa gttctgtatc tgtaagcagt atcatccaaa gtagtaccca ttgctgtaga
      601 tatgtcttgt gtgtaagggg gttggggtgg ttgtttgtgt tcttgacttt tgtgttagca
      661 agggaagacg ggcaaaaaag tgagtgtggt tgggagggag agacgagcct tatatataat
      721 gcttgtttgt gtttgtgcaa gtggacgccg aaacgggcag gagccaaact aaacaaggca
      781 gacaatgcga gcttaattgg attgcctgat gggcaggggt tagggctcga tcaatggggg
      841 tgcgaagtga caaaattggg aattaggttc gcaagcaagg ctgacaagac tttggcccaa
      901 acatttgtac gcggtggaca acaggagcca cccatcgtct gtcacgggct agccggtcgt
      961 gcgtcctgtc aggctccacc taggctccat gccactccat acaatcccac tagtgtaccg
     1021 ctaggccgct tttagctccc atctaagacc cccccaaaac ctccactgta cagtgcactg
     1081 tactgtgtgg cgatcaaggg caagggaaaa aaggcgcaaa catgcatgca tggaatgacg
     1141 taggtaaggc gttactagac tgaaaagtgg cacatttcgg cgtgccaaag ggtcctaggt
     1201 gcgtttcgcg agctgggcgc caggccaagc cgctccaaaa cgcctctccg actccctcca
     1261 gcggcctcca tatccccatc cctctccaca gcaatgttgt taagccttgc aaacgaaaaa
     1321 atagaaaggc taataagctt ccaatattgt ggtgtacgct gcataacgca acaatgagcg
     1381 ccaaacaaca cacacacaca gcacacagca gcattaacca cgatgaacag catgacatta
     1441 caggtgggtg tgtaatcagg gccctgattg ctggtggtgg gagcccccat catgggctct
     1501 cgtccagccg cggaaaccgg aaacgctcga atctgccgcc gctcatactc accgaaacgt
     1561 gtggggctcg aaaaaacggg cgccaaactc cttatactac tgtatattca agcaagtata
     1621 tccgtgggtg cgggtgattt ggatctaagg ttcgtactca acactcacga gcagcttgcc
     1681 tatgttacat ccttttatca gacatagcgg ccgcaaattt aaataaaatg aagtgaagtt
     1741 cctatacttt ctagagaata ggaacttcta tagtgagtcg aataagggcg acacaaaatt
     1801 tattctaaat gcataataaa tactgataac atcttatagt ttgtattata ttttgtatta
     1861 tcgttgacat gtataatttt gatatcaaaa actgattttc cctttattat tttcgagatt
     1921 tattttctta attctcttta acaaactaga aatattgtat atacaaaaaa tcataaataa
     1981 tagatgaata gtttaattat aggtgttcat caatcgaaaa agcaacgtat cttatttaaa
     2041 gtgcgttgct tttttctcat ttataaggtt aaataattct catatatcaa gcaaagtgac
     2101 aggcgccctt aaatattctg acaaatgctc tttccctaaa ctccccccat aaaaaaaccc
     2161 gccgaagcgg gtttttacgt tatttgcgga ttaacgatta ctcgttatca gaaccgccca
     2221 gggggcccga gcttaagact ggccgtcgtt ttacaacaca gaaagagttt gtagaaacgc
     2281 aaaaaggcca tccgtcaggg gccttctgct tagtttgatg cctggcagtt ccctactctc
     2341 gccttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2401 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2461 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2521 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2581 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2641 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2701 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2761 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2821 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2881 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2941 ggctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3001 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3061 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3121 tttgatcttt tctacggggt ctgacgctca gtggaacgac gcgcgcgtaa ctcacgttaa
     3181 gggattttgg tcatgagctt gcgccgtccc gtcaagtcag cgtaatgctc tgcttaccaa
     3241 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3301 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagcgct
     3361 gcgatgatac cgcgagaacc acgctcaccg gctccggatt tatcagcaat aaaccagcca
     3421 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3481 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3541 gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3601 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3661 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3721 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3781 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3841 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3901 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3961 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     4021 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     4081 tgttgaatac tcatattctt cctttttcaa tattattgaa gcatttatca gggttattgt
     4141 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggtcagtgtt
     4201 acaaccaatt aaccaattct gaacattatc gcgagcccat ttatacctga atatggctca
     4261 taacacccct tgtttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa
     4321 ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg actccccatg cgagagtagg
     4381 gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgcccgg
     4441 gctaattatg gggtgtcgcc cttattcgac tctatagtga agttcctatt ctctagaaag
     4501 tataggaact tctgaagtgg ggatttaaat gcggccgcga atgcacgcga taccgtaccc
     4561 acacaaaaaa agcaccaccg actcggtgcc actttttcaa gttgataacg gactagcctt
     4621 attttaactt gctatttcta gctctaaaac cactcgccag cacttattgg tgcgccgacc
     4681 cggaatcgaa ccgggggccc atcgatggca acgatggatt ttaccactaa accatcggcg
     4741 cgtttcttgg cttttgttag gaaccaacct cgaaattact cagcagtttc cttaggttag
     4801 ggcggaaatc gagtctggat aagacgtgga acgattgata cgattacttg atctgttagc
     4861 aatgattcac tatcgcacgc attccgttgc atcaaattta gggatgccat caactttcag
     4921 ttcataatta atatcttacc aaattaggta atctgcaaaa gttcagactg tgaaatgtaa
     4981 cattttatat atcaagctct atttaatgcc tcacagtagt taacataaag agatacagaa
     5041 ttgtcgtgtc agtgtatact atccatgtgt atactctgga tatccatttg tattccatta
     5101 tctacgaatt ggcggggttc tgctcgagct gggtggcgag gttgttaagt cggtccattt
     5161 ttgtgtgtct tggttggatg aggtccaaag tggggggagt ctaccaggct ttatatacac
     5221 ctggtagttg caactttcgg tatgatcccc acgaaagata aagttcagca tgtggagagc
     5281 cgaggtagta tgcgtgtgac agagacccaa actgggggtc accgcgtgca cactggcgac
     5341 gccgctctgg aaggtcacat gaccgcagat actccgttct cccctcacgt cgattcggct
     5401 cggggacacg gtcagcgcca ttgccacgtg tccacggttc tctccactct gtagaaagtc
     5461 acttccacca agttccatga agcttattag gtttttggag aaatccgggg cggcatttct
     5521 ggggtaactt gggcaagcca gtagtggatc aattgggacc cgaattgggt ggaaaaaggt
     5581 gataacgagc cctttgggat ccccacaatt gatctaagac ctctgcttgc cccgatcttg
     5641 gccccctcag ccccaacact gttgctcttc aaactccccc atacctcggt tcagatgcag
     5701 ccttcaaaac tttaacgtac ctccgagcct gtgaaaacgt ggtcatgaaa aaacgagaga
     5761 tgtaagtccc gtcacatacc gctgcgagac caccagctgt acatacactt ggcgttccag
     5821 catgcacggt cgctcaacag gcgctccccc attggcggaa agacaagccc ccactagatt
     5881 cctgttcaag ggcaaagtct agaagcccgc cccccccagc gcccgtttcc ttgttagtgc
     5941 ggcctgcaca tgtcacctgt ctggcaggta tcgtcgctac tttgccagtc gtatcgtcgt
     6001 caacggattg cgctgcccat tcacgtgtaa actctcgacg gatatcccgg agtttcccgc
     6061 cgatcctccc gcgagccact gccacgtcgc ctccaagcct gctcgagcgt cccaaaacct
     6121 tctaaagcaa ggttttcagt ataatgttac atgcgtacac gcgtctgtac agaaaaaaaa
     6181 gaaaaatttg aaatataaat aacgttctta atactaacat aactataaaa aaataaatag
     6241 ggacctagac ttcaggttgt ctaactcctt ccttttcggt tagagcggat gtggggggag
     6301 ggcgtgaatg taagcgtg
//
