LOCUS       sequence-424301-        6960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA targeting to the intergradtion site C3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6960)
  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 6960)
  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: pCfB8856
FEATURES             Location/Qualifiers
     source          1..6960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        1863..1937
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(2001..2034)
                     /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(2241..2260)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2266..2285)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2327..2418
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2419..2487,2488..3279)
                     /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(2637..2656)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3401..3989
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3890..3909
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4143..4160
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4258..4275
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4259..4275
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4745..4778
                     /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)"
     CDS             5922..6950
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             5922..6950
                     /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 ccggtcttgc ctcctactac ctgtccatca atgacgaggt tctcacccct gcccaggtcg
       61 aagctcttat tactgagtcc aacaccggtg ttcttcccac caccaacctc aagggctctc
      121 ccaacgctgt tgcctacaac ggtgttggca tttaggcaat taacagatag tttgccggtg
      181 ataattctct taacctccca cactcctttg acataacgat ttatgtaacg aaactgaaat
      241 ttgaccagat attgttgtaa atagaaaatc tggcttgtag gtggcaaaat cccgtctttg
      301 ttcatcaatt ccctctgtga ctactcgtca tccctttatg ttcgactgtc gtatttttat
      361 tttccataca tacgcaagtg agatgcccgt gtccgaattc agcaggcttg gaggcgacgt
      421 ggcagtggct cgcgggagga tcggcgggaa actccgggat atccgtcgag agtttacacg
      481 tgaatgggca gcgcaatccg ttgacgacga tacgactggc aaagtagcga cgatacctgc
      541 cagacaggtg acatgtgcag gccgcactaa caaggaaacg ggcgctgggg ggggcgggct
      601 tctagacttt gcccttgaac aggaatctag tgggggcttg tctttccgcc aatgggggag
      661 cgcctgttga gcgaccgtgc atgctggaac gccaagtgta tgtacagctg gtggtctcgc
      721 agcggtatgt gacgggactt acatctctcg ttttttcatg accacgtttt cacaggctcg
      781 gaggtacgtt aaagttttga aggctgcatc tgaaccgagg tatgggggag tttgaagagc
      841 aacagtgttg gggctgaggg ggccaagatc ggggcaagca gaggtcttag atcaattgtg
      901 gggatcccaa agggctcgtt atcacctttt tccacccaat tcgggtccca attgatccac
      961 tactggcttg cccaagttac cccagaaatg ccgccccgga tttctccaaa aacctaataa
     1021 gcttcatgga acttggtgga agtgactttc tacagagtgg agagaaccgt ggacacgtgg
     1081 caatggcgct gaccgtgtcc ccgagccgaa tcgacgtgag gggagaacgg agtatctgcg
     1141 gtcatgtgac cttccagagc ggcgtcgcca gtgtgcacgc ggtgaccccc agtttgggtc
     1201 tctgtcacac gcatactacc tcggctctcc acatgctgaa ctttatcttt cgtggggatc
     1261 ataccgaaag ttgcaactac caggtgtata taaagcctgg tagactcccc ccactttgga
     1321 cctcatccaa ccaagacaca caaaaatgga ccgacttaac aacctcgcca cccagctcga
     1381 gcagaacccc gccaattcgt agataatgga atacaaatgg atatccagag tatacacatg
     1441 gatagtatac actgacacga caattctgta tctctttatg ttaactactg tgaggcatta
     1501 aatagagctt gatatataaa atgttacatt tcacagtctg aacttttgca gattacctaa
     1561 tttggtaaga tattaattat gaactgaaag ttgatggcat ccctaaattt gatgcaacgg
     1621 aatgcgtgcg atagtgaatc attgctaaca gatcaagtaa tcgtatcaat cgttccacgt
     1681 cttatccaga ctcgatttcc gccctaacct aaggaaactg ctgagtaatt tcgaggttgg
     1741 ttcctaacaa aagccaagaa acgcgccgat ggtttagtgg taaaatccat cgttgccatc
     1801 gatgggcccc cggttcgatt ccgggtcggc gcaggttggt taaatagtgc tatggctttg
     1861 gggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     1921 gtggcaccga gtcggtggtg ctttttttgt gtgggtacgg tatcgcgtgc attcgcggcc
     1981 gcatttaaat ccccacttca gaagttccta tactttctag agaataggaa cttcactata
     2041 gagtcgaata agggcgacac cccataatta gcccgggcga aaggcccagt ctttcgactg
     2101 agcctttcgt tttatttgat gcctggcagt tccctactct cgcatgggga gtccccacac
     2161 taccatcggc gctacggcgt ttcacttctg agttcggcat ggggtcaggt gggaccaccg
     2221 cgctactgcc gccaggcaaa caaggggtgt tatgagccat attcaggtat aaatgggctc
     2281 gcgataatgt tcagaattgg ttaattggtt gtaacactga cccctatttg tttatttttc
     2341 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2401 tattgaaaaa ggaagaatat gagtattcaa catttccgtg tcgcccttat tccctttttt
     2461 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     2521 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     2581 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2641 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     2701 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     2761 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     2821 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     2881 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     2941 gagcgtgaca ccacgatgcc tgtagcgatg gcaacaacgt tgcgcaaact attaactggc
     3001 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3061 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatccgga
     3121 gccggtgagc gtggttctcg cggtatcatc gcagcgctgg ggccagatgg taagccctcc
     3181 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     3241 atcgctgaga taggtgcctc actgattaag cattggtaag cagagcatta cgctgacttg
     3301 acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgc gcgtcgttcc
     3361 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3421 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3481 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3541 atactgttct tctagtgtag ccgtagttag cccaccactt caagaactct gtagcaccgc
     3601 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3661 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3721 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3781 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3841 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3901 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3961 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4021 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4081 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4141 gcagcgagtc agtgagcgag gaagcggaag gcgagagtag ggaactgcca ggcatcaaac
     4201 taagcagaag gcccctgacg gatggccttt ttgcgtttct acaaactctt tctgtgttgt
     4261 aaaacgacgg ccagtcttaa gctcgggccc cctgggcggt tctgataacg agtaatcgtt
     4321 aatccgcaaa taacgtaaaa acccgcttcg gcgggttttt ttatgggggg agtttaggga
     4381 aagagcattt gtcagaatat ttaagggcgc ctgtcacttt gcttgatata tgagaattat
     4441 ttaaccttat aaatgagaaa aaagcaacgc actttaaata agatacgttg ctttttcgat
     4501 tgatgaacac ctataattaa actattcatc tattatttat gattttttgt atatacaata
     4561 tttctagttt gttaaagaga attaagaaaa taaatctcga aaataataaa gggaaaatca
     4621 gtttttgata tcaaaattat acatgtcaac gataatacaa aatataatac aaactataag
     4681 atgttatcag tatttattat gcatttagaa taaattttgt gtcgccctta ttcgactcac
     4741 tatagaagtt cctattctct agaaagtata ggaacttcac ttcattttat ttaaatttgc
     4801 ggccgctatg tctgataaaa ggatgtaaca taggcaagct gctcgtgagt gttgagtacg
     4861 aaccttagat ccaaatcacc cgcacccacg gatatacttg cttgaatata cagtagtata
     4921 aggagtttgg cgcccgtttt ttcgagcccc acacgtttcg gtgagtatga gcggcggcag
     4981 attcgagcgt ttccggtttc cgcggctgga cgagagccca tgatgggggc tcccaccacc
     5041 agcaatcagg gccctgatta cacacccacc tgtaatgtca tgctgttcat cgtggttaat
     5101 gctgctgtgt gctgtgtgtg tgtgttgttt ggcgctcatt gttgcgttat gcagcgtaca
     5161 ccacaatatt ggaagcttat tagcctttct attttttcgt ttgcaaggct taacaacatt
     5221 gctgtggaga gggatgggga tatggaggcc gctggaggga gtcggagagg cgttttggag
     5281 cggcttggcc tggcgcccag ctcgcgaaac gcacctagga ccctttggca cgccgaaatg
     5341 tgccactttt cagtctagta acgccttacc tacgtcattc catgcatgca tgtttgcgcc
     5401 ttttttccct tgcccttgat cgccacacag tacagtgcac tgtacagtgg aggttttggg
     5461 ggggtcttag atgggagcta aaagcggcct agcggtacac tagtgggatt gtatggagtg
     5521 gcatggagcc taggtggagc ctgacaggac gcacgaccgg ctagcccgtg acagacgatg
     5581 ggtggctcct gttgtccacc gcgtacaaat gtttgggcca aagtcttgtc agccttgctt
     5641 gcgaacctaa ttcccaattt tgtcacttcg cacccccatt gatcgagccc taacccctgc
     5701 ccatcaggca atccaattaa gctcgcattg tctgccttgt ttagtttggc tcctgcccgt
     5761 ttcggcgtcc acttgcacaa acacaaacaa gcattatata taaggctcgt ctctccctcc
     5821 caaccacact cacttttttg cccgtcttcc cttgctaaca caaaagtcaa gaacacaaac
     5881 aaccacccca acccccttac acacaagaca tatctacagc aatgggtaaa aagcctgaac
     5941 tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga
     6001 tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat
     6061 atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc
     6121 actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga
     6181 gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa
     6241 ccgaactgcc cgctgttctg cagccggtcg cggaggcaat ggatgccatt gctgcggccg
     6301 atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta
     6361 catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga
     6421 tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg
     6481 aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga
     6541 cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc
     6601 aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga
     6661 cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata
     6721 tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg
     6781 cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc
     6841 gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg
     6901 ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaataa ccccacgttg
//
