LOCUS       pBS-SKII-3XHA-fC        4839 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 4839)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2022 from SnapGene 6.1.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4839
                     /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..2577
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        1458..1801
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1802..2374
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      2380..2577
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        complement(2651..2669)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2690..2706)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2714..2730
                     /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(2738..2768)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2783..2804
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3092..3680)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3851..4711)
                     /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(4712..4816)
                     /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 catgggtacc actcttgacg acacggctta ccggtaccgc accagtgtcc cgggggacgc
     1861 cgaggccatc gaggcactgg atgggtcctt caccaccgac accgtcttcc gcgtcaccgc
     1921 caccggggac ggcttcaccc tgcgggaggt gccggtggac ccgcccctga ccaaggtgtt
     1981 ccccgacgac gaatcggacg acgaatcgga cgacggggag gacggcgacc cggactcccg
     2041 gacgttcgtc gcgtacgggg acgacggcga cctggcgggc ttcgtggtcg tctcgtactc
     2101 cggctggaac cgccggctga ccgtcgagga catcgaggtc gccccggagc accgggggca
     2161 cggggtcggg cgcgcgttga tggggctcgc gacggagttc gcccgcgagc ggggcgccgg
     2221 gcacctctgg ctggaggtca ccaacgtcaa cgcaccggcg atccacgcgt accggcggat
     2281 ggggttcacc ctctgcggcc tggacaccgc cctgtacgac ggcaccgcct cggacggcga
     2341 gcaggcgctc tacatgagca tgccctgccc ctaatcagta ctgacaataa aaagattctt
     2401 gttttcaaga acttgtcatt tgtatagttt ttttatattg tagttgttct attttaatca
     2461 aatgttagcg tgatttatat tttttttcgc ctcgacatca tctgcccaga tgcgaagtta
     2521 agtgcgcaga aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc tatactgctg
     2581 tcgattcgat actaacgccg ccactagttc tagagcggcc gccaccgcgg tggagctcca
     2641 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     2701 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     2761 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     2821 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     2881 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2941 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3001 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3061 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3121 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3181 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3241 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3301 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3361 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3421 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3481 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     3541 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3601 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3661 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3721 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3781 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3841 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3901 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3961 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4021 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4081 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4141 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4201 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4261 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4321 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4381 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4441 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4501 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4561 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4621 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4681 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4741 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4801 aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
