LOCUS       sequence-318013-        6376 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 6376)
  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 6376)
  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..6376
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             328..339
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             346..1059
                     /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      1071..1142
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1158..1185
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            1436..3010
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     CDS             1780..2808
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1780..2808
                     /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      2813..3010
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3917..4732)
                     /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(3917..4732)
                     /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     4640..4659
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4724..4743
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4957..5545
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5446..5465
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcgccttcca gacctttttt tggtttgttt ctctctccgt ggaactcgct ttctttttcg
       61 ccggtgatct cgaaacgtaa tcgatatgta ttccattcca ttcgctattt tttttgttcc
      121 ataaaccatg ttcttatgaa cagaattcgc atctagactg atgagacgaa cgtgagaccg
      181 aaagtgaaac gtgatttcat gcgtcatttt gaacattttg taaatcttat ttaataatgt
      241 gtgcggcaat tcacatttaa tttatgaatg ttttcttaac atcgcggcaa ctcaagaaac
      301 ggcaggttcg gatcttagct actagagaaa gaggagaaat actagatgcg taaaggcgaa
      361 gagctgttca ctggtgtcgt ccctattctg gtggaactgg atggtgatgt caacggtcat
      421 aagttttccg tgcgtggcga gggtgaaggt gacgcaacta atggtaaact gacgctgaag
      481 ttcatctgta ctactggtaa actgccggtt ccttggccga ctctggtaac gacgctgact
      541 tatggtgttc agtgctttgc tcgttatccg gaccatatga agcagcatga cttcttcaag
      601 tccgccatgc cggaaggcta tgtgcaggaa cgcacgattt cctttaagga tgacggcacg
      661 tacaaaacgc gtgcggaagt gaaatttgaa ggcgataccc tggtaaaccg cattgagctg
      721 aaaggcattg actttaaaga ggacggcaat atcctgggcc ataagctgga atacaatttt
      781 aacagccaca atgtttacat caccgccgat aaacaaaaaa atggcattaa agcgaatttt
      841 aaaattcgcc acaacgtgga ggatggcagc gtgcagctgg ctgatcacta ccagcaaaac
      901 actccaatcg gtgatggtcc tgttctgctg ccagacaatc actatctgag cacgcaaagc
      961 gttctgtcta aagatccgaa cgagaaacgc gatcatatgg ttctgctgga gttcgtaacc
     1021 gcagcgggca tcacgcatgg tatggatgaa ctgtacaaat gaccaggcat caaataaaac
     1081 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     1141 tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat aggtctcagc
     1201 tggaaatctg ctcgtcagtg gtgctcacac tgacgaatca tgtacagatc ataccgatga
     1261 ctgcctggcg actcacaact aagcaagaca gccggaacca gcgccggcga acaccactgc
     1321 atatatggca tatcacaaca gtccacgtct caagcagtta cagagatgtt acgaaccact
     1381 agtgcactgc agtacagttt agcttgcctc gtccccgccg ggtcacccgg ccagcgacat
     1441 ggaggcccag aataccctcc ttgacagtct tgacgtgcgc agctcagggg catgatgtga
     1501 ctgtcgcccg tacatttagc ccatacatcc ccatgtataa tcatttgcat ccatacattt
     1561 tgatggccgc acggcgcgaa gcaaaaatta cggctcctcg ctccagacct gcgagcaggg
     1621 aaacgctccc ctcacagacg cgttgaattg tccccacgcc gcgcccctgt agagaaatat
     1681 aaaaggttag gatttgccac tgaggttctt ctttcatata cttcctttta aaatcttgct
     1741 aggatacagt tctcacatca catccgaaca taaacaaaaa tgggtaaaaa gcctgaactc
     1801 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtgtc cgacctgatg
     1861 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat
     1921 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac
     1981 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt tagcgagagc
     2041 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc
     2101 gaactgcccg ctgttctgca accggtcgcg gaggccatgg atgcgatcgc tgcggccgat
     2161 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca
     2221 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg
     2281 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag
     2341 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg
     2401 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa
     2461 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg
     2521 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg
     2581 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca
     2641 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt
     2701 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc
     2761 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaataaag taactgacaa
     2821 taaaaagatt cttgttttca agaacttgtc atttgtatag tttttttata ttgtagttgt
     2881 tctattttaa tcaaatgtta gcgtgattta tatttttttt cgcctcgaca tcatctgccc
     2941 agatgcgaag ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt
     3001 cgctatactg gagtttcaca atggagggag tgaaatctaa aagtggaaaa aaaaagggga
     3061 ggcgaaaaac aaaaaaaaaa aaaagaacga accgaactat gtccacagga gcagaagcag
     3121 aagctgcggc agcagcttct gcggctttga aagaggaaac gcgtagatga aagaatgatc
     3181 caagcccggc tttccctctg tgcgcggatt gttatttttt tagacttatt tctatgtttg
     3241 ttttggacct aatataactt agtcatgaat cctggttgac tcgcgcgcat ctttttccac
     3301 tctgcgatga ttcacagtgc agcgtgcagc tcagctccag cagcagtacc agcggcagct
     3361 ccaacagtgg agccttcctt tcacgaacgg aatagaaaca ggaaggaaaa aaatagacca
     3421 acaagaggac ccatccgcga cttctaattt ttggcgcatg aaccgcgctg ttctacccgc
     3481 ccgtccgtcc tcatccctcc cccccaggaa aacaggaaat tgggaaacct aggtgcgtgc
     3541 attttcgttt tgggcgatga ttgacaatga gacttagtcc gcttggttgg gtttcgtttc
     3601 aaaaacaagg tttggaagaa gatcttggga gagaaagacc gagagatctt gaccagggcg
     3661 aagacgacca gtagcttttg gtatagccgg gataagcttc aggcacccta gggttgtata
     3721 agagttgtat gcaaggatgt gcgcaagggt ttctgaagca ggatggaaat gtggtgtgtt
     3781 tttttgtaga gcaattgcct taaagtcggg gattacgccg agacagcagc ttgacggttc
     3841 cgtggtaata tgtgtccggc aacttggcgc gcctcagccg agcggccgcg tgttacaacc
     3901 aattaaccaa ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     3961 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     4021 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     4081 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     4141 caccatgagt gacgactgaa tccggtgaga atggcaaaag cttatgcatt tctttccaga
     4201 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     4261 tattcattcg tgattgcgcc tgagcgaggc gaaatacgcg atcgctgtta aaaggacaat
     4321 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     4381 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     4441 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     4501 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     4561 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     4621 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     4681 tggaatttaa tcgcggcctg gagcaagacg tttcccgttg aatatggctc ataacacccc
     4741 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt
     4801 gtgcaatgta acatcagaga ttttgagaca caacgtggct ttgttgaata aatcgaactt
     4861 ttgctgagtt gaaggatcag tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4921 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4981 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5041 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     5101 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5161 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5221 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5281 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5341 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5401 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5461 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5521 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5581 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5641 ccgtgcggcc gcccctttca ggcgcgccgg tgttgcatta gtatcgccgt ggatgaagat
     5701 gaaggtggaa tagacgttac ccatagatac ccgaatgagt atctgattga ggatttttga
     5761 cttacttact tcttcgagat acactactct gccgaaattt cggactggaa tgcgtcaaag
     5821 tgaatccact ttgcaggaaa acgcaggaaa aacggattcg aaaacgcaac aaaaaaaaaa
     5881 aaattataca taataaaatt tgaatcaaag caacaagttc acgaatccat taaagcagtc
     5941 agaatccagg aaaaaaaaga aacagagcca gaaaaaaggc agcaacgaat cacaaccaca
     6001 gcgtattgcg tcagtggaaa ttaattacct tggtatttcc aatgctgtgt acaaccaaaa
     6061 tttccacata gttagttgat tcgcatttcc ctggtaaccg ccctcggcaa cctccctcgg
     6121 aaacttccct ccagagtaac ttcccccaga gttatagaga tatagagtta tagtagagat
     6181 atagagctat agatagattg attcttgttg tagcatttat tccgttaaac ttctgcggct
     6241 aattttgcct agcttggcct tttttttttg tttttcgagc tttcaaagac aaatggacat
     6301 tcttacataa tatattatca tatacaccga gatcggtggg tcagagcctg tagggatgga
     6361 cattttcctt ctagac
//
