LOCUS       pBS-SKII-3XHA-fC        5076 bp DNA     circular SYN 20-AUG-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5076)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2022 from SnapGene 6.1.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5076
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             735..824
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             855..1400
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylcytosine 
                     derivatives"
                     /label=CLIP-tag(TM)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLIQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISESHLAALVGNPAATAAVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLA
                     VKEWLLAHEGHRLGKPGLG"
     gene            1458..2814
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1458..1801
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1802..2611
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      2617..2814
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        complement(2888..2906)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2927..2943)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2951..2967
                     /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(2975..3005)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3020..3041
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3329..3917)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4088..4948)
                     /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"
     promoter        complement(4949..5053)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttgatatc gggtcgacgg atccccgggt
      721 taattaacat cttttaccca tacgatgttc ctgactatgc gggctatccc tatgacgtcc
      781 cggactatgc aggatcctat ccatatgacg ttccagatta cgctgctcag tgcggttcag
      841 gtggatctgg ttctatggac aaagactgcg aaatgaagcg caccaccctg gatagccctc
      901 tgggcaagct ggaactgtct gggtgcgaac agggcctgca ccgtatcatc ttcctgggca
      961 aaggaacatc tgccgccgac gccgtggaag tgcctgcccc agccgccgtg ctgggcggac
     1021 cagagccact gatccaggcc accgcctggc tcaacgccta ctttcaccag cctgaggcca
     1081 tcgaggagtt ccctgtgcca gccctgcacc acccagtgtt ccagcaggag agctttaccc
     1141 gccaggtgct gtggaaactg ctgaaagtgg tgaagttcgg agaggtcatc agcgagagcc
     1201 acctggccgc cctggtgggc aatcccgccg ccaccgccgc cgtgaacacc gccctggacg
     1261 gaaatcccgt gcccattctg atcccctgcc accgggtggt gcagggcgac agcgacgtgg
     1321 ggccctacct gggcgggctc gccgtgaaag agtggctgct ggcccacgag ggccacagac
     1381 tgggcaagcc tgggctgggt taatatataa ctgtctagaa ataaagagta tcatctttca
     1441 aaggtcaccc ggccagcgac atggaggccc agaataccct ccttgacagt cttgacgtgc
     1501 gcagctcagg ggcatgatgt gactgtcgcc cgtacattta gcccatacat ccccatgtat
     1561 aatcatttgc atccatacat tttgatggcc gcacggcgcg aagcaaaaat tacggctcct
     1621 cgctgcagac ctgcgagcag ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg
     1681 ccgcgcccct gtagagaaat ataaaaggtt aggatttgcc actgaggttc ttctttcata
     1741 tacttccttt taaaatcttg ctaggataca gttctcacat cacatccgaa cataaacaac
     1801 catgggtaag gaaaagactc acgtttcgag gccgcgatta aattccaaca tggatgctga
     1861 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     1921 attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     1981 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     2041 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     2101 cggcaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     2161 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     2221 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     2281 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     2341 gcataagctt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     2401 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     2461 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     2521 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     2581 gtttcatttg atgctcgatg agtttttcta atcagtactg acaataaaaa gattcttgtt
     2641 ttcaagaact tgtcatttgt atagtttttt tatattgtag ttgttctatt ttaatcaaat
     2701 gttagcgtga tttatatttt ttttcgcctc gacatcatct gcccagatgc gaagttaagt
     2761 gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg
     2821 attcgatact aacgccgcca ctagttctag agcggccgcc accgcggtgg agctccagct
     2881 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc
     2941 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3001 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3061 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3121 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3181 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3241 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3301 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3361 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3421 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3481 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3541 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3601 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3661 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3721 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     3781 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3841 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3901 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3961 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4021 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4081 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4141 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4201 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4261 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4321 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4381 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4441 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4501 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4561 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4621 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4681 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4741 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4801 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4861 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     4921 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     4981 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5041 taggggttcc gcgcacattt ccccgaaaag tgccac
//
