LOCUS       sequence-380554-        7807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High copy vector backbone with hygromycin B selectivity, encodes for
            TDH3(pr) driven expression of mNeon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7807)
  AUTHORS   Smith JR, Sislak CD, Fernandez Mendoza P, Carmichael L, Lewis AG, 
            Chen A, Jiang GZ, Gibney PA
  TITLE     Auxotrophy-Independent Plasmid Shuttle Vectors for Applications in 
            Diverse Yeasts
  JOURNAL   Appl Microbiol. 2024; 4(1):453-469. doi: 
            10.3390/applmicrobiol4010031
REFERENCE   2  (bases 1 to 7807)
  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: "Appl 
            Microbiol."; date: "2024"; volume: " 4(1):453-469. doi"; pages: " 
            10.3390/applmicrobiol401003"
COMMENT     Sequence Label: pRSII42H-TDH3pr-mNeon
FEATURES             Location/Qualifiers
     source          1..7807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      883..1080
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      complement(1168..1623)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1305..1326)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1517..1536
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1749..1771
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1763..1780
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1764..1780
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1790..1809
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1790..1808
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(1827..2074)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     2040..2058
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(2808..3452)
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     complement(2892..2915)
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     complement(3477..3497)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3477..3495)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3516..3532)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3516..3532)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3529..3551)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3540..3556
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3564..3570,3571..3588,3589..3594))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3609..3630
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3747..3764)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3918..4506)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3998..4017)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4677..5468,5469..5537))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5300..5319
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5538..5642)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5669..7015
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     primer_bind     7056..7074
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     7234..7253
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            join(7312..7807,1..1080)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        7312..7655
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             join(7656..7807,1..877)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             join(7656..7807,1..877)
                     /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"
ORIGIN
        1 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
       61 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
      121 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
      181 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
      241 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
      301 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
      361 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
      421 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
      481 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
      541 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
      601 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
      661 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
      721 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
      781 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
      841 ccgacgcccc agcactcgtc cgagggcaaa ggaataatca gtactgacaa taaaaagatt
      901 cttgttttca agaacttgtc atttgtatag tttttttata ttgtagttgt tctattttaa
      961 tcaaatgtta gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag
     1021 ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg
     1081 ctgtcgattc gatactaacg ccgccatcca gtgtctatgc ggtgtgaaat accgcacaga
     1141 tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt taatattttg ttaaaattcg
     1201 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     1261 cttataaatc gaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     1321 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     1381 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     1441 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     1501 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     1561 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     1621 cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     1681 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     1741 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc
     1801 actatagggc gaattgggta ccggccgcaa attaaagcct tcgagcgtcc caaaaccttc
     1861 tcaagcaagg ttttcagtat aatgttacat gcgtacacgc gtctgtacag aaaaaaaaga
     1921 aaaatttgaa atataaataa cgttcttaat actaacataa ctataaaaaa ataaataggg
     1981 acctagactt caggttgtct aactccttcc ttttcggtta gagcggatgt ggggggaggg
     2041 cgtgaatgta agcgtgacat aactaattac atgactcgag ttacttatat aattcatcca
     2101 ttcccatgac atcggtgaag gctttttgcc attccttaaa gttcaactcg gtcttactat
     2161 gttttagttc agtcttccta aacacgtaca tgggttgatt tttaagataa ttagcggcca
     2221 ttggtttagc gaatgtatac gtagtacggg cagtggatct gtatctttta ccattgccag
     2281 tggtataaga ccacttaaaa gttgaaatta ttgttttatc gttaggatat gtctttttag
     2341 acctacacca gtcagcagct gtcaggctat tagtcataac gggaccatca gccggaaaac
     2401 cagtaccctt aacttgggct tctcccttaa tatgggaacc ctcgtacgtg tacctgtagt
     2461 ttactgttaa ggaagcgccg tcttcaaact gcatggttct atgaacttgg tagccagaac
     2521 cgtctaccat agcggcttga aatggactca ttccatcagg gtaaggtaaa tattgatgaa
     2581 agccgtaccc aatatggggc acaagaatcc agggggaaaa ttgcaaatcg ccttttgttg
     2641 atttaagatt aagttcctcg tatccatcgt ttggattacc agtgccttgt ccaaccatat
     2701 caaagtcaac accattaata cttccgaaaa tatgtaattc gtgagttgct ggcaaggatg
     2761 ccatgttgtc ctcctcccct tttgagacca tactagttct agatccgtcg aaactaagtt
     2821 cttggtgttt taaaactaaa aaaaagacta actataaaag tagaatttaa gaagtttaag
     2881 aaatagattt acagaattac aatcaatacc taccgtcttt atatacttat tagtcaagta
     2941 ggggaataat ttcagggaac tggtttcaac cttttttttc agctttttcc aaatcagaga
     3001 gagcagaagg taatagaagg tgtaagaaaa tgagatagat acatgcgtgg gtcaattgcc
     3061 ttgtgtcatc atttactcca ggcaggttgc atcactccat tgaggttgtg cccgtttttt
     3121 gcctgtttgt gcccctgttc tctgtagttg cgctaagaga atggacctat gaactgatgg
     3181 ttggtgaaga aaacaatatt ttggtgctgg gattcttttt ttttctggat gccagcttaa
     3241 aaagcgggct ccattatatt tagtggatgc caggaataaa ctgttcaccc agacacctac
     3301 gatgttatat attctgtgta acccgccccc tattttgggc atgtacgggt tacagcagaa
     3361 ttaaaaggct aattttttga ctaaataaag ttaggaaaat cactactatt aattatttac
     3421 gtattctttg aaatggcgag tattgataat gataaactga gctccagctt ttgttccctt
     3481 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3541 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3601 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3661 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3721 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3781 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3841 gataacgcag gaaagaacat gtgagcaaaa ggccagcgaa aggccaggaa ccgtaaaaag
     3901 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3961 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4021 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4081 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4141 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4201 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4261 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4321 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4381 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4441 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4501 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4561 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4621 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4681 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4741 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4801 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4861 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4921 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4981 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5041 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5101 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5161 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5221 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5281 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5341 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5401 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5461 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5521 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5581 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5641 cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt ttgtagaaca
     5701 aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca tttttacaga
     5761 acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt catttttgta
     5821 aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt
     5881 acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta tacttctttt
     5941 ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc ttagattact
     6001 ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc actgtaggtc
     6061 cgttaaggtt aggagaaggc tactttggtg tctattttct cttccataaa aaaagcctga
     6121 ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt tcaagataaa
     6181 ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga acagaaggtg
     6241 atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct attttgtctc
     6301 tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca ctctatgaat
     6361 agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat aaaaaatgta
     6421 gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt tatataggga
     6481 tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg aagcggtatt
     6541 cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga aagtgcgtct
     6601 tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta gctagagaat
     6661 aggaacttcg gaataggaac ttcaaagcgt ttccgaaaac gagcgcttcc gaaaatgcaa
     6721 cgcgagctgc gcacatgcag ctcactgttc acgtcgcacc tatatctgcg tgttgcctgt
     6781 atatatatat acatgagaag aacggcatag tgcgtgttta tgcttaaatg cgtacttata
     6841 tgcgtctatt tatgtaggat gaaaggtagt ctagtacctc ctgtgatatt atcccattcc
     6901 atgcggggta tcgtatgctt ccttcagcac taccctttag ctgttctata tgctgccact
     6961 cctcaattgg attagtctca tccttcaatg ctatcatttc ctttgatatt ggatcatact
     7021 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     7081 gtctcgcgcg tttcggtgat gacggtgaaa acctctgacg catgcagctc ccggagacgg
     7141 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     7201 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
     7261 tgcaccatac caccttagct tgcctcgtcc ccgccgggtc acccggccag cgacatggag
     7321 gcccagaata ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt
     7381 cgcccgtaca tttagcccat acatccccat gtataatcat ttgcatccat acattttgat
     7441 ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc ggacctgcga gcagggaaac
     7501 gctcccctca cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa
     7561 ggttaggatt tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga
     7621 tacagttctc acatcacatc cgaacataaa caaccatggg taaaaagcct gaactcaccg
     7681 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc
     7741 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc
     7801 tgcgggt
//
