LOCUS       sequence-380561-        6974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low 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 6974)
  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 6974)
  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-mNeon
FEATURES             Location/Qualifiers
     source          1..6974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      883..1080
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      complement(1195..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
     misc_feature    5679..6182
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     6223..6241
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6279..6301)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6401..6420
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            join(6479..6974,1..1080)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        6479..6822
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             join(6823..6974,1..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
                     GNFDDAAWAQGRCDAIVRSEAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     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 tacactacgt 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 ttctggagac cgtggttggc ttgtatggag cagcagacgc gctacttcga
      601 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
      661 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
      721 gggtcgatgc gacgcaatcg tccgatccga 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 cgttaaactt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     1261 cttataaatc aaaagaatag 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 ctacagggtg
     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 taaagcctga
     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 ggccagcaaa 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 taaaaatgat 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 gccacctggg tccttttcat cacgtgctat aaaaataatt
     5701 ataatttaaa ttttttaata taaatatata aattaaaaat agaaagtaaa aaaggaaatt
     5761 aaagaaaaaa tagtttttgt tttccgaaga tgtaaaagac tctaggggga tcgccaacaa
     5821 atactacctt ttatcttgct cttcctgctc tcaggtatta atgccgaatt gtttcatctt
     5881 gtctgtgtag aagaccacac acgaaaatcc tgtgatttta cattttactt atcgttaatc
     5941 gaatgtatat ctatttaatc tgcttttctt gtctaataaa tatatatgta aagtacgctt
     6001 tttgttgaaa ttttttaaac ctttgtttat ttttttttct tcattccgta actcttctac
     6061 cttctttatt tactttctaa aatccaaata caaaacataa aaataaataa acacagagta
     6121 aattcccaaa ttattccatc attaaaagat acgaggcgcg tgtaagttac aggcaagcga
     6181 tccgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     6241 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     6301 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     6361 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
     6421 ctgagagtgc accataccac cttagcttgc ctcgtccccg ccgggtcacc cggccagcga
     6481 catggaggcc cagaataccc tccttgacag tcttgacgtg cgcagctcag gggcatgatg
     6541 tgactgtcgc ccgtacattt agcccataca tccccatgta taatcatttg catccataca
     6601 ttttgatggc cgcacggcgc gaagcaaaaa ttacggctcc tcgctgcgga cctgcgagca
     6661 gggaaacgct cccctcacag acgcgttgaa ttgtccccac gccgcgcccc tgtagagaaa
     6721 tataaaaggt taggatttgc cactgaggtt cttctttcat atacttcctt ttaaaatctt
     6781 gctaggatac agttctcaca tcacatccga acataaacaa ccatgggtaa aaagcctgaa
     6841 ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg
     6901 atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga
     6961 tatgtcctgc gggt
//
