LOCUS       sequence-317859-        6432 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting the LAC4 locus. 
            Hygromycin selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6432)
  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 6432)
  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: pI1-MTU-DO-HPH
FEATURES             Location/Qualifiers
     source          1..6432
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1127..1138
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1145..1858
                     /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      1870..1941
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1957..1984
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            2235..3809
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     CDS             2579..3607
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2579..3607
                     /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      3612..3809
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4744..5559)
                     /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(4744..5559)
                     /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     5467..5486
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5551..5570
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5784..6372
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6273..6292
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgtg cggccgcccc tttcaggcgc
       61 gccgaaagtg attgaagaac cctcaaactt gctggatatt tccttaccct tgacctttag
      121 gctttcaatt ttacccaaca atttgtccaa gacaaattgc aatccactgg attcaactga
      181 gacataacgt ttaccgtcgt tgatcttggt aggtttttct gctgtctctg taacaaaatc
      241 tggtaccttt aatggaagtt cggcttggcc ccaggcaatt tcatgacctg cctttagaac
      301 accagcatca tcctttaaca cggcaacaac ataagtggta tcagaaggaa tagtaacaga
      361 ttcttctggc tttaaagctg gaacgtcgat tgtctttccc gtgtccttgt cgataaacaa
      421 taagtgatct gtcgtaatga agtcgtgctt atttgtgatt gttacagatc cgtgcgcaat
      481 tttaatatga acgggttcaa taaccttctt atactctaca aggcccggag taggattatg
      541 ctcactgtta cacaaaccat ccatgatgaa cactccgtca tgaacctctt ccttaaagtc
      601 accaccataa gcataagctt tatgcaactt accatctgca gtactaacat cttcgaattc
      661 aataccgtga tttgcccatt cccagataaa gccaccttgg taaaacttct ccttgtagaa
      721 caactcttga tattctttca aagaacccgg gccgttaccc attgcatgac cgtactcaca
      781 caagatcaaa ggcttttcaa acttaccatt ttcatcagtg tggttcttcc tccacctttc
      841 cataatttca aatgttgggt acatgaaact aaagatatct gcactcaaag cgttcaagtc
      901 accctcataa tgcaccagtc tggtaggatc caattgctta agaattcgca tctagactga
      961 tgagacgaac gtgagaccga aagtgaaacg tgatttcatg cgtcattttg aacattttgt
     1021 aaatcttatt taataatgtg tgcggcaatt cacatttaat ttatgaatgt tttcttaaca
     1081 tcgcggcaac tcaagaaacg gcaggttcgg atcttagcta ctagagaaag aggagaaata
     1141 ctagatgcgt aaaggcgaag agctgttcac tggtgtcgtc cctattctgg tggaactgga
     1201 tggtgatgtc aacggtcata agttttccgt gcgtggcgag ggtgaaggtg acgcaactaa
     1261 tggtaaactg acgctgaagt tcatctgtac tactggtaaa ctgccggttc cttggccgac
     1321 tctggtaacg acgctgactt atggtgttca gtgctttgct cgttatccgg accatatgaa
     1381 gcagcatgac ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac gcacgatttc
     1441 ctttaaggat gacggcacgt acaaaacgcg tgcggaagtg aaatttgaag gcgataccct
     1501 ggtaaaccgc attgagctga aaggcattga ctttaaagag gacggcaata tcctgggcca
     1561 taagctggaa tacaatttta acagccacaa tgtttacatc accgccgata aacaaaaaaa
     1621 tggcattaaa gcgaatttta aaattcgcca caacgtggag gatggcagcg tgcagctggc
     1681 tgatcactac cagcaaaaca ctccaatcgg tgatggtcct gttctgctgc cagacaatca
     1741 ctatctgagc acgcaaagcg ttctgtctaa agatccgaac gagaaacgcg atcatatggt
     1801 tctgctggag ttcgtaaccg cagcgggcat cacgcatggt atggatgaac tgtacaaatg
     1861 accaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     1921 tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc
     1981 tgcgtttata ggtctcagct ggaaatctgc tcgtcagtgg tgctcacact gacgaatcat
     2041 gtacagatca taccgatgac tgcctggcga ctcacaacta agcaagacag ccggaaccag
     2101 cgccggcgaa caccactgca tatatggcat atcacaacag tccacgtctc aagcagttac
     2161 agagatgtta cgaaccacta gtgcactgca gtacagttta gcttgcctcg tccccgccgg
     2221 gtcacccggc cagcgacatg gaggcccaga ataccctcct tgacagtctt gacgtgcgca
     2281 gctcaggggc atgatgtgac tgtcgcccgt acatttagcc catacatccc catgtataat
     2341 catttgcatc catacatttt gatggccgca cggcgcgaag caaaaattac ggctcctcgc
     2401 tccagacctg cgagcaggga aacgctcccc tcacagacgc gttgaattgt ccccacgccg
     2461 cgcccctgta gagaaatata aaaggttagg atttgccact gaggttcttc tttcatatac
     2521 ttccttttaa aatcttgcta ggatacagtt ctcacatcac atccgaacat aaacaaaaat
     2581 gggtaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
     2641 cagcgtgtcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
     2701 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
     2761 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat
     2821 tggggaattt agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
     2881 gcaagacctg cctgaaaccg aactgcccgc tgttctgcaa ccggtcgcgg aggccatgga
     2941 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
     3001 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
     3061 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
     3121 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
     3181 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
     3241 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
     3301 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
     3361 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
     3421 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
     3481 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg
     3541 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
     3601 ggaataaagt aactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt
     3661 ttttttatat tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc
     3721 gcctcgacat catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca
     3781 atcgtatgtg aatgctggtc gctatactgg agttgcttaa ttagcttgta catggctttg
     3841 tggtttctgc cataacaagc ttcgttaccc aaggaccaga taataatcga aggatgattg
     3901 acatctctta ggacaagttg ggaagctctg tctaagtacg cgacctcgta ctctggatta
     3961 tctgataagt aatgggcatt aacatcgtag agtttatttt tggtatctgg atattcagcc
     4021 tccaagttcg tatgacgatt aaatggctct tgaacaccgt gagtttcaag atctgcctca
     4081 tcaatgaccc agaagcctag tttatcgaag aggtcataca ccttaggatg gtttggataa
     4141 tgcgagttac gaacagcatt gatgttaaac ttcttcatta gaatcaagtc tctaacaaca
     4201 aaatctaatg gcacagctct accgaacctt ggatggtgat catgcctgtt gacacctcta
     4261 aagagaatgt ctttgccatt aacagtaatg ttaccgtcct tcaactccac ttgtctgaaa
     4321 ccaacatggt gcttaataga ttgaatcaca ctgccatcag atccaattaa atccaactgg
     4381 tacttgtaca aagtaggatt ttctgcggtc caatgttctg gggccttgac gttgatcttg
     4441 aaagctgttt cttcgtcctt tttggtcgaa aaggaaataa attctttagt tgaaaacgtc
     4501 gtgttcccat tctcctcgtt caacaaagag cttgcatcgt aaactttaga tccatcttca
     4561 ggttcgtaaa gtgtgaaatt gatgtgatca taagaagaac cctggacatc aactttcaca
     4621 gaaagctctg catcctgata ctgagagtcc acaaaagttg tagtgaccct aacgtcttca
     4681 atatgggcct tcggcgcgcc tcagccgagc ggccgcgtgt tacaaccaat taaccaattc
     4741 tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     4801 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     4861 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     4921 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     4981 gactgaatcc ggtgagaatg gcaaaagctt atgcatttct ttccagactt gttcaacagg
     5041 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     5101 ttgcgcctga gcgaggcgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     5161 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     5221 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     5281 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     5341 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     5401 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     5461 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     5521 cggcctggag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     5581 tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca
     5641 tcagagattt tgagacacaa cgtggctttg ttgaataaat cgaacttttg ctgagttgaa
     5701 ggatcagtca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5761 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5821 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5881 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5941 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6001 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6061 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6121 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6181 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6241 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6301 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6361 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6421 tttgctcaca tg
//
