LOCUS       sequence-380553-        7589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High copy vector backbone with G418 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 7589)
  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 7589)
  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: pRSII42K-TDH3pr-mNeon
FEATURES             Location/Qualifiers
     source          1..7589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      725..922
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      complement(1010..1465)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1147..1168)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1359..1378
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1591..1613
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1605..1622
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1606..1622
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1632..1651
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1632..1650
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(1669..1916)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     1882..1900
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(2651..3295)
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     complement(2735..2758)
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     complement(3320..3340)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3320..3338)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3359..3375)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3359..3375)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3372..3394)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3383..3399
                     /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(3407..3413,3414..3431,3432..3437))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3452..3473
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3590..3607)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3761..4349)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3841..3860)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4520..5311,5312..5380))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQRFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5143..5162
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5381..5485)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5512..6858
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     primer_bind     6899..6917
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6955..6977)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7077..7096
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            join(7155..7589,1..922)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        7155..7498
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             join(7499..7589,1..719)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPICNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGDFFPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(7566..7585)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt
       61 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
      121 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
      181 atgcatggtt actcaccact gcgatccccg gcaaaacagc attccaggta ttagaagaat
      241 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
      301 cgattcctat ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
      361 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
      421 ggcctgttga acaagtctgg aaagaaatgc ataagctttt gccattctca ccggattcag
      481 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
      541 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
      601 ggaactgcct cggtgatttt tttccttcat tacagaaacg gctttttcaa aaatatggta
      661 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gcttgatgag tttttctaat
      721 cagtactgac aataaaaaga ttcttgtttt caagaacttg tcatttgtat agttttttta
      781 tattgtagtt gttctatttt aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga
      841 catcatctgc ccagatgcga agttaagtgc gcagaaagta atatcatgcg tcaatcgtat
      901 gtgaatgctg gtcgctatac tgctgtcgat tcgatactaa cgccgccatc cagtgtctat
      961 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     1021 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     1081 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     1141 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     1201 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     1261 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     1321 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     1381 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     1441 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     1501 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     1561 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     1621 gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccggccgc aaattaaagc
     1681 cttcgagcgt cccaaaacct tctcaagcaa ggttttcagt ataatgttac atgcgtacac
     1741 gcgtctgtac agaaaaaaaa gaaaaatttg aaatataaat aacgttctta atactaacat
     1801 aactataaaa aaataaatag ggacctagac ttcaggttgt ctaactcctt ccttttcggt
     1861 tagagcggat gtggggggag ggcgtgaatg taagcgtgac ataactaatt acatgactcg
     1921 agttacttat ataattcatc cattcccatg acatcggtga aggctttttg ccattcctta
     1981 aagttcaact cggtcttact atgttttagt tcagtcttcc taaacacgta catgggttga
     2041 tttttaagat aattagcggc cattggttta gcgaatgtat acgtagtacg ggcagtggat
     2101 ctgtatcttt taccattgcc agtggtataa gaccacttaa aagttgaaat tattgtttta
     2161 tcgttaggat atgtcttttt agacctacac cagtcagcag ctgtcaggct attagtcata
     2221 acgggaccat cagccggaaa accagtaccc ttaacttggg cttctccctt aatatgggaa
     2281 ccctcgtacg tgtacctgta gtttactgtt aaggaagcgc cgtcttcaaa ctgcatggtt
     2341 ctatgaactt ggtagccaga accgtctacc atagcggctt gaaatggact cattccatca
     2401 gggtaaggta aatattgatg aaagccgtac ccaatatggg gcacaagaat ccagggggaa
     2461 aattgcaaat cgccttttgt tgatttaaga ttaagttcct cgtatccatc gtttggatta
     2521 ccagtgcctt gtccaaccat atcaaagtca acaccattaa tacttccgaa aatatgtaat
     2581 tcgtgagttg ctggcaagga tgccatgttg tcctcctccc cttttgagac catactagtt
     2641 ctagaatccg tcgaaactaa gttcttggtg ttttaaaact aaaaaaaaga ctaactataa
     2701 aagtagaatt taagaagttt aagaaataga tttacagaat tacaatcaat acctaccgtc
     2761 tttatatact tattagtcaa gtaggggaat aatttcaggg aactggtttc aacctttttt
     2821 ttcagctttt tccaaatcag agagagcaga aggtaataga aggtgtaaga aaatgagata
     2881 gatacatgcg tgggtcaatt gccttgtgtc atcatttact ccaggcaggt tgcatcactc
     2941 cattgaggtt gtgcccgttt tttgcctgtt tgtgcccctg ttctctgtag ttgcgctaag
     3001 agaatggacc tatgaactga tggttggtga agaaaacaat attttggtgc tgggattctt
     3061 tttttttctg gatgccagct taaaaagcgg gctccattat atttagtgga tgccaggaat
     3121 aaactgttca cccagacacc tacgatgtta tatattctgt gtaacccgcc ccctattttg
     3181 ggcatgtacg ggttacagca gaattaaaag gctaattttt tgactaaata aagttaggaa
     3241 aatcactact attaattatt tacgtattct ttgaaatggc gagtattgat aatgataaac
     3301 tgagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt
     3361 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3421 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3481 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3541 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3601 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3661 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3721 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3781 ctgacgagca tcacaaaaat ggacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3841 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3901 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3961 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4021 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4081 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4141 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4201 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4261 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4321 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4381 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4441 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4501 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4561 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4621 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4681 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4741 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4801 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4861 cgtttggtat ggcttcattc agctccggct cccaacgatc aaggcgagtt acatgatccc
     4921 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4981 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5041 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5101 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5161 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5221 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5281 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5341 aaaagggaat aagggcgaca cggaaacgtt gaatactcat actcttcctt tttcaatatt
     5401 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5461 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaacgaagca
     5521 tctgtgcttc attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag
     5581 aatctgagct gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga
     5641 agaatctgtg cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa
     5701 caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgagagcg ctattttacc
     5761 aacaaagaat ctatacttct tttttgttct acaaaaatgc atcccgagag cgctattttt
     5821 ctaacaaagc atcttagatt actttttttc tcctttgtgc gctctataat gcagtctctt
     5881 gataactttt tgcactgtag gtccgttaag gttagaagaa ggctactttg gtgtctattt
     5941 tctcttccat aaaaaaagcc tgactccact tcccgcgttt actgattact agcgaagctg
     6001 cgggtgcatt ttttcaagat aaaggcatcc ccgattatat tctataccga tgtggattgc
     6061 gcatactttg tgaacagaaa gtgatagcgt tgatgattct tcattggtca gaaaattatg
     6121 aacggtttct tctattttgt ctctatatac tacgtatagg aaatgtttac attttcgtat
     6181 tgttttcgat tcactctatg aatagttctt actacaattt ttttgtctaa agagtaatac
     6241 tagagataaa cataaaaaat gtagaggtcg agtttagatg caagttcaag gagcgaaagg
     6301 tggatgggta ggttatatag ggatatagca cagagatata tagcaaagag atacttttga
     6361 gcaatgtttg tggaagcggt attcgcaata ttttagtagc tcgttacagt ccggtgcgtt
     6421 tttggttttt tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt
     6481 cctatacttt ctagctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa
     6541 aacgagcgct tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc
     6601 acctatatct gcgtgttgcc tgtatatata tatacgtgag aagaacggca tagtgcgtgt
     6661 ttatgcttaa atgcgtactt atatgcgtct atttatgtag gatgaaaggt agtctagtac
     6721 ctcctgtgat attatcccat tccatgcggg gtatcgtatg cttccttcag cactaccctt
     6781 tagctgttct atatgctgcc actcctcaat tggattagtc tcatccttca atgctatcat
     6841 ttcctttgat attggatcat actaagaaac cattattatc atgacattaa cctataaaaa
     6901 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     6961 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     7021 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     7081 atcagagcag attgtactga gagtgcacca taccacctta gcttgcctcg tccccgccgg
     7141 gtcacccggc cagcgacatg gaggcccaga ataccctcct tgacagtctt gacgtgcgca
     7201 gctcaggggc atgatgtgac tgtcgcccgt acatttagcc catacatccc catgtataat
     7261 catttgcatc catacatttt gatggccgca cggcgcgaag caaaaattac ggctcctcgc
     7321 tgcagacctg cgagcaggga aacgctcccc tcacagacgc gttgaattgt ccccacgccg
     7381 cgcccctgta gagaaatata aaaggttagg atttgccact gaggttcttc tttcatatac
     7441 ttccttttaa aatcttgcta ggatacagtt ctcacatcac atccgaacat aaacaaccat
     7501 gggtaaggaa aagactcacg tttcgaggcc gcgattaaat tccaacatgg atgctgattt
     7561 atatgggtat aaatgggctc gcgataatg
//
