LOCUS       sequence-424055-        4893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  integration vector for three gene cassette with integration site E4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4893)
  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 4893)
  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: pCfB12716
FEATURES             Location/Qualifiers
     source          1..4893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             709..720
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             727..1440
                     /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"
     terminator      1452..1523
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1539..1566
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    complement(2109..2142)
                     /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(2349..2368)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2374..2393)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2435..2526
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2527..2595,2596..3387)
                     /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(2745..2764)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3509..4097
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3998..4017
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4251..4268
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4366..4383
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4367..4383
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4853..4886
                     /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)"
ORIGIN
        1 tttatttaaa tttgcggccg cgaatgcgtg cgatgcggag gagcaataga catacgattt
       61 gacattttga catttcacag tatcggcagc cttgctatag caagtgtgta tttgtagata
      121 gagacaggca attagatttg gaggggaccg caagaaacag atcatcattc tgtcggcccc
      181 tgttgtccct gtggctctgt tagtccgctg gtccgtggcc ggagcttgtc atgtttctgg
      241 aataccctct ggccacatta actccgctaa agcctcaagg ccgtaaaggc gccagtggca
      301 tgttgcaagc aaagccgcac gcataacacg ccacatcacg tgagcggtca taggccggtc
      361 gccagacggc tacaacccta ttacgcgggt gctctggcca aacagaccgt ggaggaactc
      421 ttcgacactt gatattaaac tctatgccac ttatcgggcc tactgatcag actcaaggtc
      481 tattgttctg agagaggaag tttggggaga aagaacgggc acatgtgttg atctccaaac
      541 gtgagaccac tgatgagacg gaaagtgaaa cgtgatttca tgcgtcattt tgaacatttt
      601 gtaaatctta tttaataatg tgtgcggcaa ttcacattta atttatgaat gttttcttaa
      661 catcgcggca actcaagaaa cggcaggttc ggatcttagc tactagagaa agaggagaaa
      721 tactagatgc gtaaaggcga agagctgttc actggtgtcg tccctattct ggtggaactg
      781 gatggtgatg tcaacggtca taagttttcc gtgcgtggcg agggtgaagg tgacgcaact
      841 aatggtaaac tgacgctgaa gttcatctgt actactggta aactgccggt tccttggccg
      901 actctggtaa cgacgctgac ttatggtgtt cagtgctttg ctcgttatcc ggaccatatg
      961 aagcagcatg acttcttcaa gtccgccatg ccggaaggct atgtgcagga acgcacgatt
     1021 tcctttaagg atgacggcac gtacaaaacg cgtgcggaag tgaaatttga aggcgatacc
     1081 ctggtaaacc gcattgagct gaaaggcatt gactttaaag aggacggcaa tatcctgggc
     1141 cataagctgg aatacaattt taacagccac aatgtttaca tcaccgccga taaacaaaaa
     1201 aatggcatta aagcgaattt taaaattcgc cacaacgtgg aggatggcag cgtgcagctg
     1261 gctgatcact accagcaaaa cactccaatc ggtgatggtc ctgttctgct gccagacaat
     1321 cactatctga gcacgcaaag cgttctgtct aaagatccga acgagaaacg cgatcatatg
     1381 gttctgctgg agttcgtaac cgcagcgggc atcacgcatg gtatggatga actgtacaaa
     1441 tgaccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     1501 gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt
     1561 tctgcgttta tacgtctcag ttcggtctca gctgaacagg gaacatcgac tctgagacag
     1621 aggtgacctg acgagcaacg tatgcgagca actcgtatct gcagttggtt ggcctggtca
     1681 ctcgattaat tggccatccg aaagcaccat tagcctatct aacgcactaa aatggtgggg
     1741 aagtcaaagg accatggttc gttcattgtt ggctaacggc ccgaaatccg ccaaagtgtg
     1801 ggaaaaaaac ataaaaaata tataaaaaaa agttgctttg gattttggga tgcagcaacc
     1861 aaaaactcgc gcaaaagccc gctcgagtgt ctctgaacgc aaatacgacc gacgcggtct
     1921 ccaacatctc gtcgcggtcc cctcccccct tcagccagcg tccctccaga cccaggccac
     1981 agcataatat ttcagagcgt tgacacccag agcgggggag ctcgtaataa ggtacgcctg
     2041 tgcagctggg cagtacggtc cagtgttaaa tcgcgtgcat tcgcggccgc atttaaatcc
     2101 ccacttcaga agttcctata ctttctagag aataggaact tcactataga gtcgaataag
     2161 ggcgacaccc cataattagc ccgggcgaaa ggcccagtct ttcgactgag cctttcgttt
     2221 tatttgatgc ctggcagttc cctactctcg catggggagt ccccacacta ccatcggcgc
     2281 tacggcgttt cacttctgag ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc
     2341 caggcaaaca aggggtgtta tgagccatat tcaggtataa atgggctcgc gataatgttc
     2401 agaattggtt aattggttgt aacactgacc cctatttgtt tatttttcta aatacattca
     2461 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2521 aagaatatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     2581 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2641 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2701 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     2761 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     2821 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     2881 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     2941 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     3001 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     3061 acgatgcctg tagcgatggc aacaacgttg cgcaaactat taactggcga actacttact
     3121 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     3181 ctgcgctcgg cccttccggc tggctggttt attgctgata aatccggagc cggtgagcgt
     3241 ggttctcgcg gtatcatcgc agcgctgggg ccagatggta agccctcccg tatcgtagtt
     3301 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     3361 ggtgcctcac tgattaagca ttggtaagca gagcattacg ctgacttgac gggacggcgc
     3421 aagctcatga ccaaaatccc ttaacgtgag ttacgcgcgc gtcgttccac tgagcgtcag
     3481 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3541 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3601 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     3661 tagtgtagcc gtagttagcc caccacttca agaactctgt agcaccgcct acatacctcg
     3721 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3781 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3841 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3901 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3961 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4021 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4081 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4141 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4201 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4261 tgagcgagga agcggaaggc gagagtaggg aactgccagg catcaaacta agcagaaggc
     4321 ccctgacgga tggccttttt gcgtttctac aaactctttc tgtgttgtaa aacgacggcc
     4381 agtcttaagc tcgggccccc tgggcggttc tgataacgag taatcgttaa tccgcaaata
     4441 acgtaaaaac ccgcttcggc gggttttttt atggggggag tttagggaaa gagcatttgt
     4501 cagaatattt aagggcgcct gtcactttgc ttgatatatg agaattattt aaccttataa
     4561 atgagaaaaa agcaacgcac tttaaataag atacgttgct ttttcgattg atgaacacct
     4621 ataattaaac tattcatcta ttatttatga ttttttgtat atacaatatt tctagtttgt
     4681 taaagagaat taagaaaata aatctcgaaa ataataaagg gaaaatcagt ttttgatatc
     4741 aaaattatac atgtcaacga taatacaaaa tataatacaa actataagat gttatcagta
     4801 tttattatgc atttagaata aattttgtgt cgcccttatt cgactcacta tagaagttcc
     4861 tattctctag aaagtatagg aacttcactt cat
//
