LOCUS       sequence-317863-        6669 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I2 
            described in (Rajkumar et al.,2019). Hygromycin selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6669)
  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 6669)
  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: pI2-MTU-DO-HPH
FEATURES             Location/Qualifiers
     source          1..6669
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      717..788
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      804..831
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            1082..2656
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     CDS             1426..2454
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1426..2454
                     /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      2459..2656
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3707..4522)
                     /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(3707..4522)
                     /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     4430..4449
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4514..4533
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4747..5335
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5236..5255
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             6643..6654
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(6661..6669,1..705)
                     /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"
ORIGIN
        1 ggcgaagagc tgttcactgg tgtcgtccct attctggtgg aactggatgg tgatgtcaac
       61 ggtcataagt tttccgtgcg tggcgagggt gaaggtgacg caactaatgg taaactgacg
      121 ctgaagttca tctgtactac tggtaaactg ccggttcctt ggccgactct ggtaacgacg
      181 ctgacttatg gtgttcagtg ctttgctcgt tatccggacc atatgaagca gcatgacttc
      241 ttcaagtccg ccatgccgga aggctatgtg caggaacgca cgatttcctt taaggatgac
      301 ggcacgtaca aaacgcgtgc ggaagtgaaa tttgaaggcg ataccctggt aaaccgcatt
      361 gagctgaaag gcattgactt taaagaggac ggcaatatcc tgggccataa gctggaatac
      421 aattttaaca gccacaatgt ttacatcacc gccgataaac aaaaaaatgg cattaaagcg
      481 aattttaaaa ttcgccacaa cgtggaggat ggcagcgtgc agctggctga tcactaccag
      541 caaaacactc caatcggtga tggtcctgtt ctgctgccag acaatcacta tctgagcacg
      601 caaagcgttc tgtctaaaga tccgaacgag aaacgcgatc atatggttct gctggagttc
      661 gtaaccgcag cgggcatcac gcatggtatg gatgaactgt acaaatgacc aggcatcaaa
      721 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
      781 acgctctcta ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttataggt
      841 ctcagctgga aatctgctcg tcagtggtgc tcacactgac gaatcatgta cagatcatac
      901 cgatgactgc ctggcgactc acaactaagc aagacagccg gaaccagcgc cggcgaacac
      961 cactgcatat atggcatatc acaacagtcc acgtctcaag cagttacaga gatgttacga
     1021 accactagtg cactgcagta cagtttagct tgcctcgtcc ccgccgggtc acccggccag
     1081 cgacatggag gcccagaata ccctccttga cagtcttgac gtgcgcagct caggggcatg
     1141 atgtgactgt cgcccgtaca tttagcccat acatccccat gtataatcat ttgcatccat
     1201 acattttgat ggccgcacgg cgcgaagcaa aaattacggc tcctcgctcc agacctgcga
     1261 gcagggaaac gctcccctca cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag
     1321 aaatataaaa ggttaggatt tgccactgag gttcttcttt catatacttc cttttaaaat
     1381 cttgctagga tacagttctc acatcacatc cgaacataaa caaaaatggg taaaaagcct
     1441 gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtgtccgac
     1501 ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt
     1561 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
     1621 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaatttagc
     1681 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
     1741 gaaaccgaac tgcccgctgt tctgcaaccg gtcgcggagg ccatggatgc gatcgctgcg
     1801 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
     1861 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
     1921 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
     1981 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
     2041 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
     2101 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
     2161 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
     2221 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
     2281 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
     2341 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
     2401 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga ataaagtaac
     2461 tgacaataaa aagattcttg ttttcaagaa cttgtcattt gtatagtttt tttatattgt
     2521 agttgttcta ttttaatcaa atgttagcgt gatttatatt ttttttcgcc tcgacatcat
     2581 ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat
     2641 gctggtcgct atactggagt tgaagcatag aagaaatgtt taaccaaaaa ctatcctttt
     2701 tatttatttt gaattttctc tctcatattt ttgtaaaacg atactttaac cacactcaag
     2761 cttttgcttg ctactcaaat acctgaaatc tttatagaca attttccttc ttattcgtaa
     2821 taataaaact atattgttct cttcctgttt ttttttttaa aaaaaaaaac tgtcaaaatc
     2881 tcccccacag aaaaataaaa acaaattata cttttgggag taaaccatga acttaaaaca
     2941 acagttcata ttcttaagta gtgccactat agttaaccag agacaattta actcctaaat
     3001 cttcatactg aagattgaca taaactaaat aatgactaca ctgttacttt tgagtcagtt
     3061 gggtcttttg gcattcgcca atgctattaa ccctatggta aagggcgaat atagaattga
     3121 acatcctaag ataggagact tgtcagaaat tgaatctgct atctctccca aatctttcta
     3181 tgaatcattt caatatggtt ccgaagcgga gttccgtaag aattgggttt cttctgatct
     3241 aacaatggaa catgtagacg aaacatcttc gaaggcgttc cctggcacat ggaagttgca
     3301 gtcaccattt ttggtcccag gattcagtga tgatctatct ttagttttgg gtagcgaaca
     3361 aactagagct gctattgccc ataggttaaa atacgatcta tcgatcaagg ataatgagaa
     3421 actagtggtg caatatgaag ttaaacttca aaaaatgctt gaatgtggcg gtgcatatat
     3481 caaattgttg aaaacctccg caactgatct ttctcagtac gaccactcgt cagatgacta
     3541 tgctgtcatc tttggcccag acttttgcca aggttatgtg gacgaaattc atttggtttt
     3601 gaagcgcgag aatccttttt ctggaaaatc tgaagacaaa ttcttattaa atgccggcgc
     3661 gcctcagccg agcggccgcg tgttacaacc aattaaccaa ttctgattag aaaaactcat
     3721 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     3781 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     3841 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     3901 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     3961 atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     4021 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgaggc
     4081 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     4141 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     4201 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     4261 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     4321 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     4381 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     4441 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctg gagcaagacg
     4501 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     4561 ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca
     4621 caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag tcatgaccaa
     4681 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4741 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4801 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4861 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     4921 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4981 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5041 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5101 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5161 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5221 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5281 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5341 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5401 tcctgcgtta tcccctgatt ctgtggataa ccgtgcggcc gcccctttca ggcgcgccac
     5461 cgaatattaa ataatctttt tttatttctt atgtacgcct gctttgtatg atacaaaatc
     5521 atcgtatcgt attcggcttc atgttatccg tcgaatttag attaccttgg ctaaggggcc
     5581 aaggtggatg attcgtatga gtatcacaat ttacaaatta caagttttgc aaagagtatg
     5641 taaactgtaa aaaagaaaac attgacaaat atacctgaaa agagtccatt ttctacattt
     5701 gagaaaaaaa ggaataaagt gattctcctc atcgctcata aaaaaatact attcatgagc
     5761 aaaattgaga aaaatggcaa tcacattcaa tactagttag atagccatat ctaaactgct
     5821 gtacgtggtt taatactagc accaacatca tttgaataat ggcgaagaaa ccaatttcga
     5881 aacattcccg tgcggcaaga agatcagaag cagccgaacc ggaggctaag gctttagaga
     5941 ctttacccag agctgaaaag acagattttg cagcaacatt aatcagaact gctaacaaaa
     6001 acgaagcttt gctcgaagcg aagatccaaa agcgtggaca aaaaaataag aagaaaaacg
     6061 cgggaagagt gagtaagcaa cttatagatg acagttcttt aacaagtagg aaagctattc
     6121 gtgccttaaa tattaccagt aggctcgatg gtaagaggga gaaagccatt catagagcta
     6181 aatatgtgca gtcagctcgt aaagctggat gggatactac aaaccaaagc attaggaaag
     6241 aattatcgac actacaaaac gcagcaaata cggtcagcga ttccgttaac aaaagcacgg
     6301 acaaggaagg gtctgcgaat actgaagatg cgatgataga agacgatcaa gatcacattg
     6361 aaatttacga cagtgctaaa agccagaagg tacaagaaaa agcagtatca accactttta
     6421 cggcatcaaa cgtatttacg tcattagagg atgagatgaa ttcgcatcta gactgatgag
     6481 acgaacgtga gaccgaaagt gaaacgtgat ttcatgcgtc attttgaaca ttttgtaaat
     6541 cttatttaat aatgtgtgcg gcaattcaca tttaatttat gaatgttttc ttaacatcgc
     6601 ggcaactcaa gaaacggcag gttcggatct tagctactag agaaagagga gaaatactag
     6661 atgcgtaaa
//
