LOCUS       sequence-423559-        6327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA targeting to the intergradtion site E3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6327)
  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 6327)
  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: pCfB6637
FEATURES             Location/Qualifiers
     source          1..6327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(1740..1773)
                     /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(2243..2260)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2243..2259)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2358..2375)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2529..3117)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2609..2628)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3239..4030,4031..4099))
                     /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     3862..3881
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4100..4191)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4233..4252
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4258..4277
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    4484..4517
                     /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(4581..4655)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     join(6316..6327,1..7)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 gcgtgacata actaattaca tgatcatgga catggcatag acatatacaa ggcttgttca
       61 ccatctgatg cagtaccatc gtataatgca gtatccaaac cacacaaggt aaaacccatt
      121 cttctatagg catgaatagc tggagcgtta acattagtaa cttccaacca caaatgacca
      181 gcacctcttt ctctagcaaa ttcagtagcc aaacccatca aagctctacc aacaccatga
      241 cctctatgtt ctggagcaac ttcaatatct tcgacggtca atcttctatt ccaaccagaa
      301 taagaaacaa caacaaaacc agccaagtca ccgtcatcac cataagcaac aaaagttcta
      361 gaatctggat caccatcttc accatcatca gattcgtcat cggattcatc atctggaaaa
      421 accttagtca atggtggatc aactggaact tctctcaaag tgaaaccatc accagtagca
      481 gtaactctga aaacagtgtc agtagtgaaa gaaccatcca aagcttcaat agcttcagca
      541 tcacctggaa cagaagttct gtatctgtaa gcagtatcat ccaaagtagt acccattgct
      601 gtagatatgt cttgtgtgta agggggttgg ggtggttgtt tgtgttcttg acttttgtgt
      661 tagcaaggga agacgggcaa aaaagtgagt gtggttggga gggagagacg agccttatat
      721 ataatgcttg tttgtgtttg tgcaagtgga cgccgaaacg ggcaggagcc aaactaaaca
      781 aggcagacaa tgcgagctta attggattgc ctgatgggca ggggttaggg ctcgatcaat
      841 gggggtgcga agtgacaaaa ttgggaatta ggttcgcaag caaggctgac aagactttgg
      901 cccaaacatt tgtacgcggt ggacaacagg agccacccat cgtctgtcac gggctagccg
      961 gtcgtgcgtc ctgtcaggct ccacctaggc tccatgccac tccatacaat cccactagtg
     1021 taccgctagg ccgcttttag ctcccatcta agaccccccc aaaacctcca ctgtacagtg
     1081 cactgtactg tgtggcgatc aagggcaagg gaaaaaaggc gcaaacatgc atgcatggaa
     1141 tgacgtaggt aaggcgttac tagactgaaa agtggcacat ttcggcgtgc caaagggtcc
     1201 taggtgcgtt tcgcgagctg ggcgccaggc caagccgctc caaaacgcct ctccgactcc
     1261 ctccagcggc ctccatatcc ccatccctct ccacagcaat gttgttaagc cttgcaaacg
     1321 aaaaaataga aaggctaata agcttccaat attgtggtgt acgctgcata acgcaacaat
     1381 gagcgccaaa caacacacac acacagcaca cagcagcatt aaccacgatg aacagcatga
     1441 cattacaggt gggtgtgtaa tcagggccct gattgctggt ggtgggagcc cccatcatgg
     1501 gctctcgtcc agccgcggaa accggaaacg ctcgaatctg ccgccgctca tactcaccga
     1561 aacgtgtggg gctcgaaaaa acgggcgcca aactccttat actactgtat attcaagcaa
     1621 gtatatccgt gggtgcgggt gatttggatc taaggttcgt actcaacact cacgagcagc
     1681 ttgcctatgt tacatccttt tatcagacat agcggccgca aatttaaata aaatgaagtg
     1741 aagttcctat actttctaga gaataggaac ttctatagtg agtcgaataa gggcgacaca
     1801 aaatttattc taaatgcata ataaatactg ataacatctt atagtttgta ttatattttg
     1861 tattatcgtt gacatgtata attttgatat caaaaactga ttttcccttt attattttcg
     1921 agatttattt tcttaattct ctttaacaaa ctagaaatat tgtatataca aaaaatcata
     1981 aataatagat gaatagttta attataggtg ttcatcaatc gaaaaagcaa cgtatcttat
     2041 ttaaagtgcg ttgctttttt ctcatttata aggttaaata attctcatat atcaagcaaa
     2101 gtgacaggcg cccttaaata ttctgacaaa tgctctttcc ctaaactccc cccataaaaa
     2161 aacccgccga agcgggtttt tacgttattt gcggattaac gattactcgt tatcagaacc
     2221 gcccaggggg cccgagctta agactggccg tcgttttaca acacagaaag agtttgtaga
     2281 aacgcaaaaa ggccatccgt caggggcctt ctgcttagtt tgatgcctgg cagttcccta
     2341 ctctcgcctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2401 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2461 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2521 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2581 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2641 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2701 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     2761 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2821 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2881 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2941 tggtgggcta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3001 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3061 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3121 gatcctttga tcttttctac ggggtctgac gctcagtgga acgacgcgcg cgtaactcac
     3181 gttaagggat tttggtcatg agcttgcgcc gtcccgtcaa gtcagcgtaa tgctctgctt
     3241 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3301 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3361 gcgctgcgat gataccgcga gaaccacgct caccggctcc ggatttatca gcaataaacc
     3421 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3481 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3541 ttgttgccat cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3601 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3661 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3721 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3781 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3841 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3901 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3961 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4021 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4081 ggaaatgttg aatactcata ttcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4141 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggtca
     4201 gtgttacaac caattaacca attctgaaca ttatcgcgag cccatttata cctgaatatg
     4261 gctcataaca ccccttgttt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg
     4321 ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggactcc ccatgcgaga
     4381 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     4441 cccgggctaa ttatggggtg tcgcccttat tcgactctat agtgaagttc ctattctcta
     4501 gaaagtatag gaacttctga agtggggatt taaatgcggc cgcgaatgca cgcgataccg
     4561 tacccacaca aaaaaagcac caccgactcg gtgccacttt ttcaagttga taacggacta
     4621 gccttatttt aacttgctat ttctagctct aaaacgacca tcgactgatt caaggtaacc
     4681 aacctgcgcc gacccggaat cgaaccgggg gcccatcgat ggcaacgatg gattttacca
     4741 ctaaaccatc ggcgcgtttc ttggcttttg ttaggaacca acctcgaaat tactcagcag
     4801 tttccttagg ttagggcgga aatcgagtct ggataagacg tggaacgatt gatacgatta
     4861 cttgatctgt tagcaatgat tcactatcgc acgcattccg ttgcatcaaa tttagggatg
     4921 ccatcaactt tcagttcata attaatatct taccaaatta ggtaatctgc aaaagttcag
     4981 actgtgaaat gtaacatttt atatatcaag ctctatttaa tgcctcacag tagttaacat
     5041 aaagagatac agaattgtcg tgtcagtgta tactatccat gtgtatactc tggatatcca
     5101 tttgtattcc attatctacg aattggcggg gttctgctcg agctgggtgg cgaggttgtt
     5161 aagtcggtcc atttttgtgt gtcttggttg gatgaggtcc aaagtggggg gagtctacca
     5221 ggctttatat acacctggta gttgcaactt tcggtatgat ccccacgaaa gataaagttc
     5281 agcatgtgga gagccgaggt agtatgcgtg tgacagagac ccaaactggg ggtcaccgcg
     5341 tgcacactgg cgacgccgct ctggaaggtc acatgaccgc agatactccg ttctcccctc
     5401 acgtcgattc ggctcgggga cacggtcagc gccattgcca cgtgtccacg gttctctcca
     5461 ctctgtagaa agtcacttcc accaagttcc atgaagctta ttaggttttt ggagaaatcc
     5521 ggggcggcat ttctggggta acttgggcaa gccagtagtg gatcaattgg gacccgaatt
     5581 gggtggaaaa aggtgataac gagccctttg ggatccccac aattgatcta agacctctgc
     5641 ttgccccgat cttggccccc tcagccccaa cactgttgct cttcaaactc ccccatacct
     5701 cggttcagat gcagccttca aaactttaac gtacctccga gcctgtgaaa acgtggtcat
     5761 gaaaaaacga gagatgtaag tcccgtcaca taccgctgcg agaccaccag ctgtacatac
     5821 acttggcgtt ccagcatgca cggtcgctca acaggcgctc ccccattggc ggaaagacaa
     5881 gcccccacta gattcctgtt caagggcaaa gtctagaagc ccgccccccc cagcgcccgt
     5941 ttccttgtta gtgcggcctg cacatgtcac ctgtctggca ggtatcgtcg ctactttgcc
     6001 agtcgtatcg tcgtcaacgg attgcgctgc ccattcacgt gtaaactctc gacggatatc
     6061 ccggagtttc ccgccgatcc tcccgcgagc cactgccacg tcgcctccaa gcctgctcga
     6121 gcgtcccaaa accttctcaa gcaaggtttt cagtataatg ttacatgcgt acacgcgtct
     6181 gtacagaaaa aaaagaaaaa tttgaaatat aaataacgtt cttaatacta acataactat
     6241 aaaaaaataa atagggacct agacttcagg ttgtctaact ccttcctttt cggttagagc
     6301 ggatgtgggg ggagggcgtg aatgtaa
//
