LOCUS       sequence-61678-e        4512 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 4512)
  TITLE     Formosa lab yeast 3' tagging constructs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4512)
  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            "Unpublished"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: pTF274 (pFA6a-PSc-6xGly-3xFlag-HphMX)
FEATURES             Location/Qualifiers
     source          1..4512
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(199..303)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     371..389
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(427..449)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     549..568
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        649..667
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     649..666
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             728..751
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             776..841
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     terminator      870..1057
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(925..949)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1132..2707
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1132..1475
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1476..2504
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1476..2504
                     /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"
     terminator      2510..2707
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2814..2833)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2815..2833)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2920..2937)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3091..3679)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3171..3190)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3850..4512,1..129,130..198))
                     /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     4473..4492
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
       61 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
      121 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
      181 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
      241 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      301 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
      361 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
      421 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
      481 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
      541 taactatgcg gcatcagagc agattgtact gagagtgcac catatggaca tattgtcgtt
      601 agaacgcggc tacaattaat acataacctt atgtatcata cacatacgat ttaggtgaca
      661 ctatagaacg cggccgccag ctgaagcttc gtacgctgca ggtcgacgga tccccgggtt
      721 aattaacttg gaagtattgt tccaaggtcc aggtggaggc ggtggaggct ctagagacta
      781 caaggaccac gacggtgact acaaggacca cgacattgac tacaaggatg acgatgacaa
      841 gtgactcgag gcgcgccact tctaaataag cgaatttctt atgatttatg atttttatta
      901 ttaaataagt tataaaaaaa ataagtgtat acaaatttta aagtgactct taggttttaa
      961 aacgaaaatt cttattcttg agtaactctt tcctgtaggt caggttgctt tctcaggtat
     1021 agtatgaggt cgctcttatt gaccacacct ctaccggcag atccgctagg gataacaggg
     1081 taatatagat ctgtttagct tgcctcgtcc ccgccgggtc acccggccag cgacatggag
     1141 gcccagaata ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt
     1201 cgcccgtaca tttagcccat acatccccat gtataatcat ttgcatccat acattttgat
     1261 ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc agacctgcga gcagggaaac
     1321 gctcccctca cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa
     1381 ggttaggatt tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga
     1441 tacagttctc acatcacatc cgaacataaa caaccatggg taaaaagcct gaactcaccg
     1501 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc
     1561 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc
     1621 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg
     1681 catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga
     1741 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac
     1801 tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta
     1861 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc
     1921 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg
     1981 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact
     2041 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca
     2101 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg
     2161 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct
     2221 acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc
     2281 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt
     2341 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac
     2401 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata
     2461 gtggaaaccg acgccccagc actcgtccga gggcaaagga ataatcagta ctgacaataa
     2521 aaagattctt gttttcaaga acttgtcatt tgtatagttt ttttatattg tagttgttct
     2581 attttaatca aatgttagcg tgatttatat tttttttcgc ctcgacatca tctgcccaga
     2641 tgcgaagtta agtgcgcaga aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc
     2701 tatactgctg tcgattcgat actaacgccg ccatccagtg tcgaaaacga gctcgaattc
     2761 atcgatgata tcagatccac tagtggccta tgcggccgcg gatctgccgg tctccctata
     2821 gtgagtcgta ttaatttcga taagccaggt taacctgcat taatgaatcg gccaacgcgc
     2881 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     2941 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3001 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3061 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3121 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3181 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3241 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag
     3301 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3361 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3421 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3481 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     3541 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3601 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3661 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3721 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3781 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3841 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3901 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3961 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     4021 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     4081 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4141 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4201 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4261 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4321 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4381 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4441 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4501 aaaagtgctc at
//
