LOCUS       sequence-69212-e        4804 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 ClonNAT resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4804)
  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 4804)
  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: pAAH6
FEATURES             Location/Qualifiers
     source          1..4804
                     /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            1440..2559
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        1440..1783
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1784..2356
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      2362..2559
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2619..2639)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2619..2637)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2658..2674)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2658..2674)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2671..2693)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2682..2698
                     /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(2706..2712,2713..2730,2731..2736))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2751..2772
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2889..2906)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3060..3648)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3140..3159)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3819..4610,4611..4679))
                     /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     4442..4461
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4680..4784)
                     /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 taagaattct gcagatatcc
     1321 atcacactgg cggccgccag ctgaagcttc gtacgctgca ggtcgacgga tccccgggtt
     1381 aattaaggcg cgccagatct gtttagcttg cctcgtcccc gccgggtcac ccggccagcg
     1441 acatggaggc ccagaatacc ctccttgaca gtcttgacgt gcgcagctca ggggcatgat
     1501 gtgactgtcg cccgtacatt tagcccatac atccccatgt ataatcattt gcatccatac
     1561 attttgatgg ccgcacggcg cgaagcaaaa attacggctc ctcgctgcag acctgcgagc
     1621 agggaaacgc tcccctcaca gacgcgttga attgtcccca cgccgcgccc ctgtagagaa
     1681 atataaaagg ttaggatttg ccactgaggt tcttctttca tatacttcct tttaaaatct
     1741 tgctaggata cagttctcac atcacatccg aacataaaca accatgggta ccactcttga
     1801 cgacacggct taccggtacc gcaccagtgt cccgggggac gccgaggcca tcgaggcact
     1861 ggatgggtcc ttcaccaccg acaccgtctt ccgcgtcacc gccaccgggg acggcttcac
     1921 cctgcgggag gtgccggtgg acccgcccct gaccaaggtg ttccccgacg acgaatcgga
     1981 cgacgaatcg gacgacgggg aggacggcga cccggactcc cggacgttcg tcgcgtacgg
     2041 ggacgacggc gacctggcgg gcttcgtggt cgtctcgtac tccggctgga accgccggct
     2101 gaccgtcgag gacatcgagg tcgccccgga gcaccggggg cacggggtcg ggcgcgcgtt
     2161 gatggggctc gcgacggagt tcgcccgcga gcggggcgcc gggcacctct ggctggaggt
     2221 caccaacgtc aacgcaccgg cgatccacgc gtaccggcgg atggggttca ccctctgcgg
     2281 cctggacacc gccctgtacg acggcaccgc ctcggacggc gagcaggcgc tctacatgag
     2341 catgccctgc ccctaatcag tactgacaat aaaaagattc ttgttttcaa gaacttgtca
     2401 tttgtatagt ttttttatat tgtagttgtt ctattttaat caaatgttag cgtgatttat
     2461 attttttttc gcctcgacat catctgccca gatgcgaagt taagtgcgca gaaagtaata
     2521 tcatgcgtca atcgtatgtg aatgctggtc gctatactgc tgtcgattcg atactaacgc
     2581 cgccatccag tgtcgaaaac gagctccagc ttttgttccc tttagtgagg gttaattgcg
     2641 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2701 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2761 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2821 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2881 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2941 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3001 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3061 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3121 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3181 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3241 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3301 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3361 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3421 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3481 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3541 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3601 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3661 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3721 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3781 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3841 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3901 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3961 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4021 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4081 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4141 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4201 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4261 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4321 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4381 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4441 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4501 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4561 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4621 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4681 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4741 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4801 gtgc
//
