LOCUS       sequence-310230-        6379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I4 
            described in (Rajkumar et al.,2019). Hygromycin selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6379)
  AUTHORS   Rajkumar AS, Morrissey JP
  TITLE     Rational engineering of Kluyveromyces marxianus to create a chassis 
            for the production of aromatic products.
  JOURNAL   Microb Cell Fact. 2020 Nov 11;19(1):207. doi: 
            10.1186/s12934-020-01461-7.
  PUBMED    33176787
REFERENCE   2  (bases 1 to 6379)
  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: "Microb Cell
            Fact."; date: "2020-11-11"; volume: "19(1):207. doi"; pages: " 
            10.1186/s12934-020-01461-7"
COMMENT     Sequence Label: pI4-MTU-DO-HPH
FEATURES             Location/Qualifiers
     source          1..6379
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1052..1063
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1070..1783
                     /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      1795..1866
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1882..1909
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            2160..3734
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     CDS             2504..3532
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2504..3532
                     /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"
     terminator      3537..3734
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4644..5459)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4644..5459)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5367..5386
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5451..5470
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5684..6272
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6173..6192
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccctttcagg cgcgccggtg ttgcattagt atcgccgtgg atgaagatga aggtggaata
       61 gacgttaccc atagataccc gaatgagtat ctgattgagg atttttgact tacttacttc
      121 ttcgagatac actactctgc cgaaatttcg gactggaatg cgtcaaagtg aatccacttt
      181 gcaggaaaac gcaggaaaaa cggattcgaa aacgcaacaa aaaaaaaaaa attatacata
      241 ataaaatttg aatcaaagca acaagttcac gaatccatta aagcagtcag aatccaggaa
      301 aaaaaagaaa cagagccaga aaaaaggcag caacgaatca caaccacagc gtattgcgtc
      361 agtggaaatt aattaccttg gtatttccaa tgctgtgtac aaccaaaatt tccacatagt
      421 tagttgattc gcatttccct ggtaaccgcc ctcggcaacc tccctcggaa acttccctcc
      481 agagtaactt cccccagagt tatagagata tagagttata gtagagatat agagctatag
      541 atagattgat tcttgttgta gcatttattc cgttaaactt ctgcggctaa ttttgcctag
      601 cttggccttt tttttttgtt tttcgagctt tcaaagacaa atggacattc ttacataata
      661 tattatcata tacaccgaga tcggtgggtc agagcctgta gggatggaca ttttccttct
      721 agactcgcct tccagacctt tttttggttt gtttctctct ccgtggaact cgctttcttt
      781 ttcgccggtg atctcgaaac gtaatcgata tgtattccat tccattcgct attttttttg
      841 ttccataaac catgttctta tgaacagaat tcgcatctag actgatgaga cgaacgtgag
      901 accgaaagtg aaacgtgatt tcatgcgtca ttttgaacat tttgtaaatc ttatttaata
      961 atgtgtgcgg caattcacat ttaatttatg aatgttttct taacatcgcg gcaactcaag
     1021 aaacggcagg ttcggatctt agctactaga gaaagaggag aaatactaga tgcgtaaagg
     1081 cgaagagctg ttcactggtg tcgtccctat tctggtggaa ctggatggtg atgtcaacgg
     1141 tcataagttt tccgtgcgtg gcgagggtga aggtgacgca actaatggta aactgacgct
     1201 gaagttcatc tgtactactg gtaaactgcc ggttccttgg ccgactctgg taacgacgct
     1261 gacttatggt gttcagtgct ttgctcgtta tccggaccat atgaagcagc atgacttctt
     1321 caagtccgcc atgccggaag gctatgtgca ggaacgcacg atttccttta aggatgacgg
     1381 cacgtacaaa acgcgtgcgg aagtgaaatt tgaaggcgat accctggtaa accgcattga
     1441 gctgaaaggc attgacttta aagaggacgg caatatcctg ggccataagc tggaatacaa
     1501 ttttaacagc cacaatgttt acatcaccgc cgataaacaa aaaaatggca ttaaagcgaa
     1561 ttttaaaatt cgccacaacg tggaggatgg cagcgtgcag ctggctgatc actaccagca
     1621 aaacactcca atcggtgatg gtcctgttct gctgccagac aatcactatc tgagcacgca
     1681 aagcgttctg tctaaagatc cgaacgagaa acgcgatcat atggttctgc tggagttcgt
     1741 aaccgcagcg ggcatcacgc atggtatgga tgaactgtac aaatgaccag gcatcaaata
     1801 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     1861 gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttataggtct
     1921 cagctggaaa tctgctcgtc agtggtgctc acactgacga atcatgtaca gatcataccg
     1981 atgactgcct ggcgactcac aactaagcaa gacagccgga accagcgccg gcgaacacca
     2041 ctgcatatat ggcatatcac aacagtccac gtctcaagca gttacagaga tgttacgaac
     2101 cactagtgca ctgcagtaca gtttagcttg cctcgtcccc gccgggtcac ccggccagcg
     2161 acatggaggc ccagaatacc ctccttgaca gtcttgacgt gcgcagctca ggggcatgat
     2221 gtgactgtcg cccgtacatt tagcccatac atccccatgt ataatcattt gcatccatac
     2281 attttgatgg ccgcacggcg cgaagcaaaa attacggctc ctcgctccag acctgcgagc
     2341 agggaaacgc tcccctcaca gacgcgttga attgtcccca cgccgcgccc ctgtagagaa
     2401 atataaaagg ttaggatttg ccactgaggt tcttctttca tatacttcct tttaaaatct
     2461 tgctaggata cagttctcac atcacatccg aacataaaca aaaatgggta aaaagcctga
     2521 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tgtccgacct
     2581 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg
     2641 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg
     2701 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aatttagcga
     2761 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga
     2821 aaccgaactg cccgctgttc tgcaaccggt cgcggaggcc atggatgcga tcgctgcggc
     2881 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac
     2941 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt
     3001 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc
     3061 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct
     3121 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc
     3181 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca
     3241 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta
     3301 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga
     3361 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg
     3421 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact
     3481 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat aaagtaactg
     3541 acaataaaaa gattcttgtt ttcaagaact tgtcatttgt atagtttttt tatattgtag
     3601 ttgttctatt ttaatcaaat gttagcgtga tttatatttt ttttcgcctc gacatcatct
     3661 gcccagatgc gaagttaagt gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc
     3721 tggtcgctat actggagttt cacaatggag ggagtgaaat ctaaaagtgg aaaaaaaaag
     3781 gggaggcgaa aaacaaaaaa aaaaaaaaga acgaaccgaa ctatgtccac aggagcagaa
     3841 gcagaagctg cggcagcagc ttctgcggct ttgaaagagg aaacgcgtag atgaaagaat
     3901 gatccaagcc cggctttccc tctgtgcgcg gattgttatt tttttagact tatttctatg
     3961 tttgttttgg acctaatata acttagtcat gaatcctggt tgactcgcgc gcatcttttt
     4021 ccactctgcg atgattcaca gtgcagcgtg cagctcagct ccagcagcag taccagcggc
     4081 agctccaaca gtggagcctt cctttcacga acggaataga aacaggaagg aaaaaaatag
     4141 accaacaaga ggacccatcc gcgacttcta atttttggcg catgaaccgc gctgttctac
     4201 ccgcccgtcc gtcctcatcc ctccccccca ggaaaacagg aaattgggaa acctaggtgc
     4261 gtgcattttc gttttgggcg atgattgaca atgagactta gtccgcttgg ttgggtttcg
     4321 tttcaaaaac aaggtttgga agaagatctt gggagagaaa gaccgagaga tcttgaccag
     4381 ggcgaagacg accagtagct tttggtatag ccgggataag cttcaggcac cctagggttg
     4441 tataagagtt gtatgcaagg atgtgcgcaa gggtttctga agcaggatgg aaatgtggtg
     4501 tgtttttttg tagagcaatt gccttaaagt cggggattac gccgagacag cagcttgacg
     4561 gttccgtggt aatatgtgtc cggcaacagg cgcgcctcag ccgaccgagc ggccgcgtgt
     4621 tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg aaactgcaat
     4681 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     4741 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     4801 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     4861 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct
     4921 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     4981 aaaccgttat tcattcgtga ttgcgcctga gcgaggcgaa atacgcgatc gctgttaaaa
     5041 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     5101 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     5161 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     5221 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     5281 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     5341 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     5401 tccatgttgg aatttaatcg cggcctggag caagacgttt cccgttgaat atggctcata
     5461 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt
     5521 ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttg ttgaataaat
     5581 cgaacttttg ctgagttgaa ggatcagtca tgaccaaaat cccttaacgt gagttttcgt
     5641 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5701 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     5761 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5821 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5881 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5941 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6001 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6061 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6121 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6181 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6241 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6301 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     6361 tggataaccg tgcggccgc
//
