LOCUS       pBS-SKII-3XHA-eD        5007 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 5007)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2022 from SnapGene 6.1.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5007
                     /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..1334
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     gene            1389..2745
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1389..1732
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1733..2542
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      2548..2745
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        complement(2819..2837)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2858..2874)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2882..2898
                     /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(2906..2936)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2951..2972
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3260..3848)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4019..4879)
                     /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(4880..4984)
                     /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 ttctatgatc agtctgattg cggcgttagc ggtagatcgc gttatcggca
      901 tggaaaacgc catgccgtgg aacctgcctg ccgatctcgc ctggtttaaa cgcaacacct
      961 taaataaacc cgtgattatg ggccgccata cctgggaatc aatcggtcgt ccgttgccgg
     1021 gacgcaaaaa tattatcctc agcagtcaac cgggtacgga cgatcgcgta acgtgggtga
     1081 agtcggtgga tgaagccatc gcggcgtgtg gtgacgtacc agaaatcatg gtgattggcg
     1141 gcggtcgcgt ttatgaacag ttcttgccaa aagcgcaaaa actgtatctg acgcatatcg
     1201 acgcagaagt ggaaggcgac acccatttcc cggattacga gccggatgac tgggaatcgg
     1261 tattcagcga attccacgat gctgatgcgc agaactctca cagctattgc tttgagattc
     1321 tggagcggcg gtaatatata actgtctaga aataaagagt atcatctttc aaaggtcacc
     1381 cggccagcga catggaggcc cagaataccc tccttgacag tcttgacgtg cgcagctcag
     1441 gggcatgatg tgactgtcgc ccgtacattt agcccataca tccccatgta taatcatttg
     1501 catccataca ttttgatggc cgcacggcgc gaagcaaaaa ttacggctcc tcgctgcaga
     1561 cctgcgagca gggaaacgct cccctcacag acgcgttgaa ttgtccccac gccgcgcccc
     1621 tgtagagaaa tataaaaggt taggatttgc cactgaggtt cttctttcat atacttcctt
     1681 ttaaaatctt gctaggatac agttctcaca tcacatccga acataaacaa ccatgggtaa
     1741 ggaaaagact cacgtttcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     1801 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
     1861 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     1921 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     1981 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggcaaaac
     2041 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     2101 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     2161 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     2221 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataagct
     2281 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     2341 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     2401 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     2461 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     2521 gatgctcgat gagtttttct aatcagtact gacaataaaa agattcttgt tttcaagaac
     2581 ttgtcatttg tatagttttt ttatattgta gttgttctat tttaatcaaa tgttagcgtg
     2641 atttatattt tttttcgcct cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa
     2701 gtaatatcat gcgtcaatcg tatgtgaatg ctggtcgcta tactgctgtc gattcgatac
     2761 taacgccgcc actagttcta gagcggccgc caccgcggtg gagctccagc ttttgttccc
     2821 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2881 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2941 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3001 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3061 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3121 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3181 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3241 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3301 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3361 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3421 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3481 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3541 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3601 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3661 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     3721 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3781 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3841 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3901 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3961 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4021 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4081 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4141 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4201 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4261 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4321 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4381 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4441 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4501 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4561 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4621 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4681 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4741 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4801 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4861 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4921 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4981 cgcgcacatt tccccgaaaa gtgccac
//
