LOCUS       sequence-69202-e        5413 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used as a PCR template for tagging yeast proteins with the SNAP tag 
            by homologous recombination and selection for hygromycin 
            resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5413)
  AUTHORS   Hoskins AA, Friedman LJ, Gallagher SS, Crawford DJ, Anderson EG, 
            Wombacher R, Ramirez N, Cornish VW, Gelles J, Moore MJ
  TITLE     Ordered and dynamic assembly of single spliceosomes.
  JOURNAL   Science. 2011 Mar 11;331(6022):1289-95.
  PUBMED    21393538
REFERENCE   2  (bases 1 to 5413)
  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: "Science."; 
            date: "2011-03-11"; volume: "331(6022)"; pages: "1289-95"
COMMENT     Sequence Label: pAAH5
FEATURES             Location/Qualifiers
     source          1..5413
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             698..720
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             728..1273
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     gene            1367..2942
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1367..1710
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1711..2739
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1711..2739
                     /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      2745..2942
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3082..3100)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3096..3143
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3228..3248)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3228..3246)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3267..3283)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3267..3283)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3280..3302)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3291..3307
                     /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(3315..3321,3322..3339,3340..3345))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3360..3381
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3498..3515)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3669..4257)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3749..3768)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4428..5219,5220..5288))
                     /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     5051..5070
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5289..5393)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
      661 cgggcccaga ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga
      721 tatacatatg gacaaagatt gcgaaatgaa acgtaccacc ctggatagcc cgctgggcaa
      781 actggaactg agcggctgcg aacagggcct gcatgaaatt aaactgctgg gtaaaggcac
      841 cagcgcggcc gatgcggttg aagttccggc cccggccgcc gtgctgggtg gtccggaacc
      901 gctgatgcag gcgaccgcgt ggctgaacgc gtattttcat cagccggaag cgattgaaga
      961 atttccggtt ccggcgctgc atcatccggt gtttcagcag gagagcttta cccgtcaggt
     1021 gctgtggaaa ctgctgaaag tggttaaatt tggcgaagtg attagctatc agcagctggc
     1081 ggccctggcg ggtaatccgg cggccaccgc cgccgttaaa accgcgctga gcggtaaccc
     1141 ggtgccgatt ctgattccgt gccatcgtgt ggttagctct agcggtgcgg ttggcggtta
     1201 tgaaggtggt ctggcggtga aagagtggct gctggcccat gaaggtcatc gtctgggtaa
     1261 accgggtctg ggacctgcag gtataggcgc gccaggatcc ccgggttaat taaggcgcgc
     1321 cagatctgtt tagcttgcct cgtccccgcc gggtcacccg gccagcgaca tggaggccca
     1381 gaataccctc cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc
     1441 gtacatttag cccatacatc cccatgtata atcatttgca tccatacatt ttgatggccg
     1501 cacggcgcga agcaaaaatt acggctcctc gctgcggacc tgcgagcagg gaaacgctcc
     1561 cctcacagac gcgttgaatt gtccccacgc cgcgcccctg tagagaaata taaaaggtta
     1621 ggatttgcca ctgaggttct tctttcatat acttcctttt aaaatcttgc taggatacag
     1681 ttctcacatc acatccgaac ataaacaacc atgggtaaaa agcctgaact caccgcgacg
     1741 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     1801 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     1861 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     1921 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     1981 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     2041 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     2101 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     2161 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     2221 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     2281 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     2341 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     2401 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     2461 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     2521 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     2581 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     2641 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     2701 aaccgacgcc ccagcactcg tccgagggca aaggaataat cagtactgac aataaaaaga
     2761 ttcttgtttt caagaacttg tcatttgtat agttttttta tattgtagtt gttctatttt
     2821 aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga catcatctgc ccagatgcga
     2881 agttaagtgc gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac
     2941 tgctgtcgat tcgatactaa cgccgccatc cagtgtcgaa aacgagctcg aattcatcga
     3001 tgatatccat cacactggcg gccgctcgag cagatccggc tgctaacaaa gcccgaaagg
     3061 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     3121 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggataa tctagaggca
     3181 ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg
     3241 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3301 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3361 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3421 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3481 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3541 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3601 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3661 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3721 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3781 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3841 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3901 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3961 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4021 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4081 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4141 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4201 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4261 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4321 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4381 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4441 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4501 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4561 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4621 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4681 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4741 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4801 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4861 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4921 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4981 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5041 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5101 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5161 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5221 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5281 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5341 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5401 ccccgaaaag tgc
//
