LOCUS       sequence-380562-        6314 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low copy vector backbone with hygromycin B selectivity, contains 
            TDH3(pr)-MCS-CYC1(3'UTR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6314)
  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 6314)
  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: pRSII41H-TDH3pr
FEATURES             Location/Qualifiers
     source          1..6314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(767..1110)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(1169..1188)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1288..1310
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1348..1366)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    1407..1910
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        1947..2051
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2052..2120,2121..2912)
                     /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     complement(2270..2289)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3083..3671
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3572..3591
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3825..3842
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3959..3980
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3995..4000,4001..4018,4019..4025)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4033..4049
                     /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)."
     primer_bind     4038..4060
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4057..4073
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4057..4073
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4092..4112
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4094..4112
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4137..4781
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     4674..4697
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     4786..4802
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4786..4802
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4836..4852)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4837..4853)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      4855..5102
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4871..4889)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5120..5139)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5121..5139)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5149..5166)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5149..5165)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5158..5180)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5306..5734
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5393..5412)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5603..5624
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     gene            complement(join(5849..6314,1..1110))
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(5849..6046)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(join(6052..6314,1..766))
                     /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
                     GNFDDAAWAQGRCDAIVRSEAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
ORIGIN
        1 ccggatgcct ccgctcgaag tagcgcgtct gctgctccat acaagccaac cacggtctcc
       61 agaagaagat gttggcgacc tcgtattggg aatccccgaa catcgcctcg ctccagtcaa
      121 tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca
      181 cgaggtgccg gacttcgggg cagtcctcgg cccaaagcat cagctcatcg agagcctgcg
      241 cgacggacgc actgacggtg tcgtccatca cagtttgcca gtgatacaca tggggatcag
      301 caatcgcgca tatgaaatca cgccacgtag tgtattgacc gattccttgc ggtccgaatg
      361 ggccgaaccc gctcgtctgg ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga
      421 ccggctgcag aacagcgggc agttcggttt caggcaggtc ttgcaacgtg acaccctgtg
      481 cacggcggga gatgcaatag gtcaggctct cgctgaattc cccaatgtca agcacttccg
      541 gaatcgggag cgcggccgat gcaaagtgcc gataaacata acgatctttg tagaaaccat
      601 cggcgcagct atttacccgc aggacatatc cacgccctcc tacatcgaag ctgaaagcac
      661 gagattcttc gccctccgag agctgcatca ggtcggagac gctgtcgaac ttttcgatca
      721 gaaacttctc gacagacgtc gcggtgagtt caggcttttt acccatggtt gtttatgttc
      781 ggatgtgatg tgagaactgt atcctagcaa gattttaaaa ggaagtatat gaaagaagaa
      841 cctcagtggc aaatcctaac cttttatatt tctctacagg ggcgcggcgt ggggacaatt
      901 caacgcgtct gtgaggggag cgtttccctg ctcgcaggtc cgcagcgagg agccgtaatt
      961 tttgcttcgc gccgtgcggc catcaaaatg tatggatgca aatgattata catggggatg
     1021 tatgggctaa atgtacgggc gacagtcaca tcatgcccct gagctgcgca cgtcaagact
     1081 gtcaaggagg gtattctggg cctccatgtc gctggccggg tgacccggcg gggacgaggc
     1141 aagctaaggt ggtatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     1201 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     1261 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     1321 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     1381 tcatgataat aatggtttct tagacggatc gcttgcctgt aacttacacg cgcctcgtat
     1441 cttttaatga tggaataatt tgggaattta ctctgtgttt atttattttt atgttttgta
     1501 tttggatttt agaaagtaaa taaagaaggt agaagagtta cggaatgaag aaaaaaaaat
     1561 aaacaaaggt ttaaaaaatt tcaacaaaaa gcgtacttta catatatatt tattagacaa
     1621 gaaaagcaga ttaaatagat atacattcga ttaacgataa gtaaaatgta aaatcacagg
     1681 attttcgtgt gtggtcttct acacagacaa gatgaaacaa ttcggcatta atacctgaga
     1741 gcaggaagag caagataaaa ggtagtattt gttggcgatc cccctagagt cttttacatc
     1801 ttcggaaaac aaaaactatt ttttctttaa tttccttttt tactttctat ttttaattta
     1861 tatatttata ttaaaaaatt taaattataa ttatttttat agcacgtgat gaaaaggacc
     1921 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1981 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2041 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     2101 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2161 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2221 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     2281 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2341 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     2401 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     2461 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     2521 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2581 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2641 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     2701 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2761 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2821 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2881 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     2941 tttagattga tttaaaacat catttttaat ttaaaaggat ctaggtgaag atcctttttg
     3001 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3061 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3121 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3181 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     3241 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3301 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3361 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3421 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3481 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3541 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3601 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3661 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3721 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3781 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3841 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     3901 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     3961 atgtgagtta gctcactcat taggcacctc aggctttaca ctttatgctt ccggctcgta
     4021 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     4081 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctc agtttatcat
     4141 tatcaatact cgccatttca aagaatacgt aaataattaa tagtagtgat tttcctaact
     4201 ttatttagtc aaaaaattag ccttttaatt ctgctgtaac ccgtacatgc ccaaaatagg
     4261 gggcgggtta cacagaatat ataacatcgt aggtgtctgg gtgaacagtt tattcctggc
     4321 atccactaaa tataatggag cccgcttttt aagctggcat ccagaaaaaa aaagaatccc
     4381 agcaccaaaa tattgttttc ttcaccaacc atcagttcat aggtccattc tcttagcgca
     4441 actacagaga acaggggcac aaacaggcaa aaaacgggca caacctcaat ggagtgatgc
     4501 aacctgcctg gagtaaatga tgacacaagg caattgaccc acgcatgtat ctatctcatt
     4561 ttcttacacc ttctattacc ttctgctctc tctgatttgg aaaaagctga aaaaaaaggt
     4621 tgaaaccagt tccctgaaat tattccccta cttgactaat aagtatataa agacggtagg
     4681 tattgattgt aattctgtaa atctatttct taaacttctt aaattctact tttatagtta
     4741 gtcttttttt tagttttaaa acaccaagaa cttagtttcg acggatctag aactagtgga
     4801 tcccccgggc tgcaggaatt cgatatcaag cttatcgata ccgtcgacct cgagtcatgt
     4861 aattagttat gtcacgctta cattcacgcc ctccccccac atccgctcta accgaaaagg
     4921 aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt atgttagtat
     4981 taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc gtgtacgcat
     5041 gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag gctttaattt
     5101 gcggccggta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     5161 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     5221 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     5281 gttgcgcagc ctgaatggcg aatggacgca ccctgtagcg gcgcattaag cgcggcgggt
     5341 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     5401 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5461 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5521 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5581 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     5641 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     5701 aatgagctga tttaacaaaa gtttaacgcg aattttaaca aaatattaac gcttacaatt
     5761 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagacactg
     5821 gatggcggcg ttagtatcga atcgacagca gtatagcgac cagcattcac atacgattga
     5881 cgcatgatat tactttctgc gcacttaact tcgcatctgg gcagatgatg tcgaggcgaa
     5941 aaaaaatata aatcacgcta acatttgatt aaaatagaac aactacaata taaaaaaact
     6001 atacaaatga caagttcttg aaaacaagaa tctttttatt gtcagtactg attattcctt
     6061 tgccctcgga cgagtgctgg ggcgtcggtt tccactatcg gcgagtactt ctacacagcc
     6121 atcggtccag acggccgcgc ttctgcgggc gatttgtgta cgcccgacag tcccggcttc
     6181 ggatcggacg attgcgtcgc atcgaccctg cgcccaagct gcatcatcga aattgccgtc
     6241 aaccaagctc tgatagagtt ggtcaagacc aatgcggagc atatacgccc ggagccgcgg
     6301 cgatcctgca agct
//
