LOCUS       sequence-318052-        6358 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I3 
            described in (Rajkumar et al.,2019). Hygromycin selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6358)
  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 6358)
  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: pI3-MTU-DO-HPH
FEATURES             Location/Qualifiers
     source          1..6358
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1038..1049
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1056..1769
                     /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      1781..1852
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1868..1895
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            2146..3720
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     CDS             2490..3518
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2490..3518
                     /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      3523..3720
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4611..5426)
                     /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(4611..5426)
                     /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     5334..5353
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5418..5437
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5651..6239
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6140..6159
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgcctcatac cgtttatgat tgcatttacg ttactgaccg gagttttgtc cgtatttgtg
       61 aagccgttgg agataattga ggcaccaagg ttcgaaccag actatgctat attctaccca
      121 ggaattaatt gccagtcatg ccatgctgtt aaagtacccc gttcgaaaca ctgttcactg
      181 tgtcggcgat gcattccatt gcaagatcat cactgcattt ggatcaacaa ttgtgttggc
      241 tacggtaact atgaatactt ctatcccttc ctactagcga actgtaccat tttatcgtat
      301 gcatccatca gactattttc aatgttgaga gttctgcgag ttgtgagcaa acatttcctg
      361 tctctctttt tgttaacaac agcgtttgca ttacttgcca taatatttac atactaccag
      421 ttaaagctcg ttcaagaagg aatgaccaac aacgagcaag ataaatggta cctcgtacat
      481 gaacatatga gggatggaaa ccttttcaag gataacaatg gctcactgta ctacagaacc
      541 agttctggtg tggaaggaat tgatgacaca ttttactcta ccaacttgta cgatcacaga
      601 aagtacaaac tcaacaatcc ttcgagaata cacagcgcgg aagaactcac aaatatttat
      661 gatagaggag acttctggaa gaaccttgta gagaggataa gtgtcctttg aaaatctgac
      721 tttaagaatt ctcgtagctc catgtacata ataaatatag actaataaga ttcgcataac
      781 aaactggctt caaatcataa tggcaaagaa tcacaaaatt cgagatttca atctagctaa
      841 tattcctaac cggaattcgc atctagactg atgagacgaa cgtgagaccg aaagtgaaac
      901 gtgatttcat gcgtcatttt gaacattttg taaatcttat ttaataatgt gtgcggcaat
      961 tcacatttaa tttatgaatg ttttcttaac atcgcggcaa ctcaagaaac ggcaggttcg
     1021 gatcttagct actagagaaa gaggagaaat actagatgcg taaaggcgaa gagctgttca
     1081 ctggtgtcgt ccctattctg gtggaactgg atggtgatgt caacggtcat aagttttccg
     1141 tgcgtggcga gggtgaaggt gacgcaacta atggtaaact gacgctgaag ttcatctgta
     1201 ctactggtaa actgccggtt ccttggccga ctctggtaac gacgctgact tatggtgttc
     1261 agtgctttgc tcgttatccg gaccatatga agcagcatga cttcttcaag tccgccatgc
     1321 cggaaggcta tgtgcaggaa cgcacgattt cctttaagga tgacggcacg tacaaaacgc
     1381 gtgcggaagt gaaatttgaa ggcgataccc tggtaaaccg cattgagctg aaaggcattg
     1441 actttaaaga ggacggcaat atcctgggcc ataagctgga atacaatttt aacagccaca
     1501 atgtttacat caccgccgat aaacaaaaaa atggcattaa agcgaatttt aaaattcgcc
     1561 acaacgtgga ggatggcagc gtgcagctgg ctgatcacta ccagcaaaac actccaatcg
     1621 gtgatggtcc tgttctgctg ccagacaatc actatctgag cacgcaaagc gttctgtcta
     1681 aagatccgaa cgagaaacgc gatcatatgg ttctgctgga gttcgtaacc gcagcgggca
     1741 tcacgcatgg tatggatgaa ctgtacaaat gaccaggcat caaataaaac gaaaggctca
     1801 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag
     1861 tcacactggc tcaccttcgg gtgggccttt ctgcgtttat aggtctcagc tggaaatctg
     1921 ctcgtcagtg gtgctcacac tgacgaatca tgtacagatc ataccgatga ctgcctggcg
     1981 actcacaact aagcaagaca gccggaacca gcgccggcga acaccactgc atatatggca
     2041 tatcacaaca gtccacgtct caagcagtta cagagatgtt acgaaccact agtgcactgc
     2101 agtacagttt agcttgcctc gtccccgccg ggtcacccgg ccagcgacat ggaggcccag
     2161 aataccctcc ttgacagtct tgacgtgcgc agctcagggg catgatgtga ctgtcgcccg
     2221 tacatttagc ccatacatcc ccatgtataa tcatttgcat ccatacattt tgatggccgc
     2281 acggcgcgaa gcaaaaatta cggctcctcg ctccagacct gcgagcaggg aaacgctccc
     2341 ctcacagacg cgttgaattg tccccacgcc gcgcccctgt agagaaatat aaaaggttag
     2401 gatttgccac tgaggttctt ctttcatata cttcctttta aaatcttgct aggatacagt
     2461 tctcacatca catccgaaca taaacaaaaa tgggtaaaaa gcctgaactc accgcgacgt
     2521 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtgtc cgacctgatg cagctctcgg
     2581 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     2641 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     2701 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt tagcgagagc ctgacctatt
     2761 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     2821 ctgttctgca accggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     2881 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     2941 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     3001 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     3061 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     3121 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     3181 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     3241 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     3301 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     3361 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     3421 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     3481 accgacgccc cagcactcgt ccgagggcaa aggaataaag taactgacaa taaaaagatt
     3541 cttgttttca agaacttgtc atttgtatag tttttttata ttgtagttgt tctattttaa
     3601 tcaaatgtta gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag
     3661 ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg
     3721 gagtaattca aggttgttca atattccgat tgttagtaga ttcagttcta ccttcgctca
     3781 gttttcttaa tgactcaaag gatattagaa ttgaaaaatt tcaaaaaagc tttaagcata
     3841 gaaccacgca cttattaaat acaatggaga aatgatttgg tgggcaatta ccggtttcaa
     3901 ttgactccga acaggttcaa atacaccaca tttaacagac tactaagaaa gagtcaaaaa
     3961 gatttggctg aacattaagg gaggctaggt tttttagtga ctttttgcga gagaaacgct
     4021 tttttagcag attaagcaat aagtttaaac atgtccgttg aattgtccaa agtatccatc
     4081 ttgggtcagg acaccattca tgtcggatac cagattcagg aacacattgt gaagactact
     4141 ctggaacaat gcaaatcgtc gacatatgtt atcatcaacg atactaatgt ctaccaggtg
     4201 ccttactacc atgacttggt tgagctattt gagaagtctc tacccgagac ttcccgtttg
     4261 ctcacatatg ttgtgaagcc aggcgaggct aacaagaccc gtgaaactaa ggcacagatt
     4321 gaagacttta tgttgaatga agggtgcact cgtgataccg tcgttattgc aattggtggt
     4381 ggtgtgatcg gtgatatgat tgggtttgtt gctgcgacat tcatgagagg tgttcgtttc
     4441 attcaaattc ctacatcgct attggcaatg gttgactcgt ctattggtgg taagactgcc
     4501 gttgacaccc caatcggtaa aaacttcatt ggtgccttct ggcaaccaca atttgtgttg
     4561 gcgcgcctca gccgagcggc cgcgtgttac aaccaattaa ccaattctga ttagaaaaac
     4621 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     4681 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     4741 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     4801 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     4861 gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca gccattacgc
     4921 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     4981 aggcgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     5041 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     5101 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     5161 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     5221 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     5281 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     5341 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctggagcaa
     5401 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     5461 agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca gagattttga
     5521 gacacaacgt ggctttgttg aataaatcga acttttgctg agttgaagga tcagtcatga
     5581 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5641 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5701 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5761 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5821 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5881 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5941 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6001 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6061 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6121 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6181 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6241 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     6301 tctttcctgc gttatcccct gattctgtgg ataaccgtgc ggccgcccct ttcaggcg
//
