LOCUS       sequence-64346-e        6991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6991)
  TITLE     Yeast Secretion Vectors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6991)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pRS314-4M5.3
FEATURES             Location/Qualifiers
     source          1..6991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    69..572
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        609..713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(714..782,783..1574)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(932..951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1745..2333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2487..2504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2657..2662,2663..2680,2681..2687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2695..2711
                     /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     2700..2722
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2719..2735
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2719..2735
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2756..2774
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2830..2848
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2880..2896
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2881..2897
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2925..3589
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3141..3258
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3553..3576
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             join(3598..3654,3655..3852)
                     /codon_start=1
                     /gene="MF-alpha-1"
                     /product="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /label=alpha-factor secretion signal
                     /note="Cleavage by the Kex2 protease occurs after the 
                     dibasic KR sequence."
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVQLDKR"
     CDS             3859..3882
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(5053..5071)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5111..5129)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5136..5152)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5145..5167)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5297..5752
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5384..5403)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5594..5615
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5849..6523)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(6524..6805)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(6822..6841)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6941..6963
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
      121 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
      181 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
      241 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
      301 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
      361 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
      421 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
      481 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
      541 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1141 cgaaggagct aaccgctttt tttcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1501 gcagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1861 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     1921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2101 cgacctacac cgaactgaga tacctacagc gtgagcattg agaaagcgcc acgcttcccg
     2161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2221 gggagcttcc aggggggaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2281 gacttgagcg tcgatttttg tgatgctcgt caggggggcc gagcctatgg aaaaacgcca
     2341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2521 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2581 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tacctcactc
     2641 attaggcacc ccaggcttta cactttatgc ttccggctcc tatgttgtgt ggaattgtga
     2701 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctcggaatta
     2761 accctcacta aagggaacaa aagctgattt acacaggaac agctatgacc atgattacgc
     2821 caagctcgaa attaaccctc actaaaggga acaaaagctg gtaccgggcc ccccctcgat
     2881 cgaggtcgac ggtatcgatg agcttgatat cgaattcctt gaattttcaa aaattcttac
     2941 tttttttttg gatggacgca aagaagttta ataatcatat tacatggcat taccaccata
     3001 tacatatcca tatacatatc catatctaat cttacttata tgttgtggaa atgtaaagag
     3061 ccccattatc ttagcctaaa aaaaccttct ctttggaact ttcagtaata cgcttaactg
     3121 ctcattgcta tattgaagta cggattagaa gccgccgagc gggtgacagc cctccgaagg
     3181 aagactctcc tccgtgcgtc ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc
     3241 tcgcgccgca ctgctccgaa caataaagat tctacaatac tagcttttat ggttatgaag
     3301 aggaaaaatt ggcagtaacc tggccccaca aaccttcaaa tgaacgaatc aaattaacaa
     3361 ccataggatg ataatgcgat tagtttttta gccttatttc tggggtaatt aatcagcgaa
     3421 gcgatgattt ttgatctatt aacagatata taaatgcaaa aactgcataa ccactttaac
     3481 taatactttc aacattttcg gtttgtatta cttcttattc aaatgtaata aaagtatcaa
     3541 caaaaaattg ttaatatacc tctatacttt aacgtcaagg agaaaaaacc cgcatgcatg
     3601 agatttcctt caatttttac tgcagtttta ttcgcagcat cctccgcatt agctgctcca
     3661 gtcaacacta caacagaaga tgaaacggca caaattccgg ctgaagctgt catcggttac
     3721 ttagatttag aaggggattt cgatgttgct gttttgccat tttccaacag cacaaataac
     3781 gggttattgt ttataaatac tactattgcc agcattgctg ctaaagaaga aggggtacag
     3841 ctggataaaa gactctttga ctacaaggac gacgatgaca aggctagcga cgtcgttatg
     3901 actcaaacac cactatcact tcctgttagt ctaggtgatc aagcctccat ctcttgcaga
     3961 tctagtcaga gcctcgtaca cagtaatgga aacacctatt tacgttggta cctgcagaag
     4021 ccaggccagt ctccaaaggt cctgatctac aaagtttcca accgagtttc tggggtccca
     4081 gacaggttca gtggcagtgg atcagggaca gatttcacac tcaagatcaa cagagtggag
     4141 gctgaggatc tgggagttta tttctgctct caaagtacac atgttccgtg gacgttcggt
     4201 ggaggcacca agcttgaaat taagtcctct gctgatgatg ctaagaagga tgctgctaag
     4261 aaggatgatg ctaagaaaga tgatgctaag aaagatggtg gcgtcaaact ggatgagact
     4321 ggaggaggct tggtgcaacc tgggggggcc atgaaactct cctgtgttac ctctggattc
     4381 acttttggtc actactggat gaactgggtc cgccagtctc cagagaaagg actggagtgg
     4441 gtagcacaat ttagaaacaa accttataat tatgaaacat attattcaga ttctgtgaaa
     4501 ggcagattca ccatctcaag agatgattcc aaaagtagtg tctatctgca aatgaacaac
     4561 ttaagagttg aagacacggg tatctattac tgtacgggtg cttcctatgg tatggaatac
     4621 ttgggtcaag gaacctcagt caccgtctcc tgataacaaa agcttatttc tgaagaagac
     4681 ttgtaatagc tcgagcgaaa tctgataaca acagtgtaga tgtaacaaaa tcgactttgt
     4741 tcccactgta cttttagctc gtacaaaata caatatactt ttcatttctc cgtaaacaac
     4801 atgttttccc atgtaatatc cttttctatt tttcgttccg ttaccaactt tacacatact
     4861 ttatatagct attcacttct atacactaaa aaactaagac aattttaatt ttgctgcctg
     4921 ccatatttca atttgttata aattcctata atttatccta ttagtagcta aaaaaagatg
     4981 aatgtgaatc gaatcctaag agaattccgg atcctctaga gcggccgcca ccgcggtgga
     5041 gctccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt tacaacgtcg
     5101 gacaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt
     5161 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccccttcgcc
     5221 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     5281 aatggcgaat ggcgcgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     5341 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     5401 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5461 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     5521 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5581 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     5641 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5701 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcctgatgc
     5761 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caggcaagtg cacaaacaat
     5821 acttaaataa atactactca gtaataacct atttcttagc atttttgacg aaatttgcta
     5881 ttttgttaga gtcttttaca ccatttgtct ccacacctcc gcttacatca acaccaataa
     5941 cgccatttaa tctaagcgca tcaccaacat tttctggcgt cagtccacca gctaacataa
     6001 aatgtaagct ttcggggctc tcttgccttc caacccagtc agaaatcgag ttccaatcca
     6061 aaagttcacc tgtcccacct gcttctgaat caaacaaggg aataaacgaa tgaggtttct
     6121 gtgaagctgc actgagtagt atgttgcagt cttttggaaa tacgagtctt ttaataactg
     6181 gcaaaccgag gaactcttgg tattcttgcc acgactcatc tccatgcagt tggacgatat
     6241 caatgccgta atcattgacc agagccaaaa catcctcctt aggttgatta cgaaacacgc
     6301 caaccaagta tttcggagtg cctgaactat ttttatatgc ttttacaaga cttgaaattt
     6361 tccttgcaat aaccgggtca attgttctct ttctattggg cacacatata atacccagca
     6421 agtcagcatc ggaatctaga gcacattctg cggcctctgt gctctgcaag ccgcaaactt
     6481 tcaccaatgg accagaacta cctgtgaaat taataacaga catactccaa gctgcctttg
     6541 tgtgcttaat cacgtatact cacgtgctca atagtcacca atgccctccc tcttggccct
     6601 ctccttttct tttttcgacc gaattaattc ttaatcggca aaaaaagaaa agctccggat
     6661 caagattgta cgtaaggtga caagctattt ttcaataaag aatatcttcc actactgcca
     6721 tctggcgtca taactgcaaa gtacacatat attacgatgc tgttctatta aatgcttcct
     6781 atattatata tatagtaatg tcgtttatgg tgcactctca gtacaatctg ctctgatgcc
     6841 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     6901 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     6961 aggttttcac cgtcatcacc gaaacgcgcg a
//
