LOCUS       sequence-428709-        4877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  integration vector for three gene cassette with integration site C3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4877)
  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 4877)
  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: pCfB12717
FEATURES             Location/Qualifiers
     source          1..4877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             289..300
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    complement(1015..1048)
                     /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(1518..1535)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1518..1534)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1633..1650)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1804..2392)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1884..1903)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2514..3305,3306..3374))
                     /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     3137..3156
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3375..3466)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3508..3527
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3533..3552
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    3759..3792
                     /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)"
     terminator      complement(4320..4347)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(4363..4434)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(4446..4877,1..282))
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
ORIGIN
        1 ctgcacatag ccttccggca tggcggactt gaagaagtca tgctgcttca tatggtccgg
       61 ataacgagca aagcactgaa caccataagt cagcgtcgtt accagagtcg gccaaggaac
      121 cggcagttta ccagtagtac agatgaactt cagcgtcagt ttaccattag ttgcgtcacc
      181 ttcaccctcg ccacgcacgg aaaacttatg accgttgaca tcaccatcca gttccaccag
      241 aatagggacg acaccagtga acagctcttc gcctttacgc atctagtatt tctcctcttt
      301 ctctagtagc taagatccga acctgccgtt tcttgagttg ccgcgatgtt aagaaaacat
      361 tcataaatta aatgtgaatt gccgcacaca ttattaaata agatttacaa aatgttcaaa
      421 atgacgcatg aaatcacgtt tcactttccg tctcatcagt ggtctcacgt ttacagtgtc
      481 tatcaacggg gcttgttcgg gccccgtaat agggtcgctt atggtggtcg tatacgggca
      541 tctcctctga caagagtgtg gaacaaccat agaagcctgc atctatctgg aaatgtggag
      601 agcaggaggg taaaaaccac ctgttcatgg gctagatccg cacgttacat gcactgagag
      661 tagactagcc gcaagaaaaa ttacgtgtgc tgtactttac ttgtaccggt aggtcttaga
      721 tgaatctatt tgccatcact gagaacaata gcggatcgag ttgaagatcc ttccccctat
      781 cgtggctcgt tctgttaacc tcttcatgga tgacccaacc tctgacccaa cctcaacctc
      841 cccactcaaa ataaaaaaaa aaaaagtatg aagatgcaca agacattgct cgcgaccact
      901 gtctgagaca atactgtatc cgcacatagc cggagcgggt tatgactcca tcaccacact
      961 actacagttt tttatcgcac gcattcgcgg ccgcaaattt aaataaaatg aagtgaagtt
     1021 cctatacttt ctagagaata ggaacttcta tagtgagtcg aataagggcg acacaaaatt
     1081 tattctaaat gcataataaa tactgataac atcttatagt ttgtattata ttttgtatta
     1141 tcgttgacat gtataatttt gatatcaaaa actgattttc cctttattat tttcgagatt
     1201 tattttctta attctcttta acaaactaga aatattgtat atacaaaaaa tcataaataa
     1261 tagatgaata gtttaattat aggtgttcat caatcgaaaa agcaacgtat cttatttaaa
     1321 gtgcgttgct tttttctcat ttataaggtt aaataattct catatatcaa gcaaagtgac
     1381 aggcgccctt aaatattctg acaaatgctc tttccctaaa ctccccccat aaaaaaaccc
     1441 gccgaagcgg gtttttacgt tatttgcgga ttaacgatta ctcgttatca gaaccgccca
     1501 gggggcccga gcttaagact ggccgtcgtt ttacaacaca gaaagagttt gtagaaacgc
     1561 aaaaaggcca tccgtcaggg gccttctgct tagtttgatg cctggcagtt ccctactctc
     1621 gccttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     1681 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     1741 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     1801 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     1861 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     1921 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     1981 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2041 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2101 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2161 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2221 ggctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     2281 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     2341 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     2401 tttgatcttt tctacggggt ctgacgctca gtggaacgac gcgcgcgtaa ctcacgttaa
     2461 gggattttgg tcatgagctt gcgccgtccc gtcaagtcag cgtaatgctc tgcttaccaa
     2521 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     2581 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagcgct
     2641 gcgatgatac cgcgagaacc acgctcaccg gctccggatt tatcagcaat aaaccagcca
     2701 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     2761 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     2821 gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     2881 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     2941 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3001 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3061 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3121 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3181 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3241 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     3301 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     3361 tgttgaatac tcatattctt cctttttcaa tattattgaa gcatttatca gggttattgt
     3421 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggtcagtgtt
     3481 acaaccaatt aaccaattct gaacattatc gcgagcccat ttatacctga atatggctca
     3541 taacacccct tgtttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa
     3601 ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg actccccatg cgagagtagg
     3661 gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgcccgg
     3721 gctaattatg gggtgtcgcc cttattcgac tctatagtga agttcctatt ctctagaaag
     3781 tataggaact tctgaagtgg ggatttaaat gcggccgcga atgcacgcga taattattgc
     3841 acaggacaca cacaaggttt catgggggga agaagaaaag aagggccaaa aaaaccaaga
     3901 cagggaacag tgacaaacaa cagccattgg ctgcttctcc gaaagcaggg tcttggaggc
     3961 ggaagctcga ggtttgtttc tggaccggcc tgagacaagc aagaggcaca catgagagaa
     4021 accatgcatt tccggtttcg ggagcgctct taccgaagtt cctttttatt ttttgttttt
     4081 gattttttat tttttggcgg gaaaaattaa tcacgaaatt atgaaattag atgccggcaa
     4141 caaaagtatt gttccaacga tataagcggc taacaaacgg ccttccgtgg acaggactag
     4201 ttttagtggc tatctaattt cgtggccgct cgctccatga tcgtcgtacg gatcgacacg
     4261 aacaatggcc actctacaat agtgctatgg ccagctgaga ccgaactgag acgtataaac
     4321 gcagaaaggc ccacccgaag gtgagccagt gtgactctag tagagagcgt tcaccgacaa
     4381 acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct
     4441 ggtcatttgt acagttcatc cataccatgc gtgatgcccg ctgcggttac gaactccagc
     4501 agaaccatat gatcgcgttt ctcgttcgga tctttagaca gaacgctttg cgtgctcaga
     4561 tagtgattgt ctggcagcag aacaggacca tcaccgattg gagtgttttg ctggtagtga
     4621 tcagccagct gcacgctgcc atcctccacg ttgtggcgaa ttttaaaatt cgctttaatg
     4681 ccattttttt gtttatcggc ggtgatgtaa acattgtggc tgttaaaatt gtattccagc
     4741 ttatggccca ggatattgcc gtcctcttta aagtcaatgc ctttcagctc aatgcggttt
     4801 accagggtat cgccttcaaa tttcacttcc gcacgcgttt tgtacgtgcc gtcatcctta
     4861 aaggaaatcg tgcgttc
//
