LOCUS       pBS-SKII-3XHA-HA        5640 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 5640)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2022 from SnapGene 6.1.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5640
                     /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..1745
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     gene            1803..3378
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1803..2146
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2147..3175
                     /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      3181..3378
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        complement(3452..3470)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3491..3507)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3515..3531
                     /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(3539..3569)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3584..3605
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3893..4481)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4652..5512)
                     /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(5513..5617)
                     /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 gggctatccg tatgacgtcc
      781 cggactatgc aggatcctat ccatatgacg ttccagatta cgctgctcag tgcggttcag
      841 gtggatctgg ttctatggca gaaatcggta ctggctttcc attcgacccg cattatgtgg
      901 aagtcctggg cgagagaatg cattacgttg acgtgggtcc gagagatgga actccggtcc
      961 tttttctgca cgggaatcct acaagctctt atgtttggcg caatatcatc cctcatgtag
     1021 ctccgacgca tcgctgtatt gcgccggacc tgattggtat gggaaaatct gataaaccag
     1081 acctgggtta ctttttcgat gatcatgtgc gtttcatgga tgccttcatt gaggcattag
     1141 ggcttgaaga agtcgtcttg gtgattcatg attggggctc agctctggga tttcactggg
     1201 ctaaaagaaa tcctgaacgc gtaaaaggca tcgcgtttat ggagttcatt cgtccaattc
     1261 cgacttggga tgaatggcct gagttcgcga gagaaacatt tcaagcattt cgcacgaccg
     1321 atgtaggccg gaagttaatc atcgatcaga atgtctttat cgaagggaca ttgccgatgg
     1381 gagtcgttcg tccgttaaca gaagtcgaaa tggatcacta tagagaacct tttcttaatc
     1441 ctgtggacag agagccgctg tggcggtttc cgaacgaact gccgattgca ggcgagcctg
     1501 ctaacattgt agcgctggtt gaagagtata tggattggct tcatcagtct ccagttccga
     1561 agttattgtt ttggggtacg cctggcgtgc ttattccacc ggccgaagcg gcacgtttgg
     1621 caaaaagcct gccaaattgc aaagccgttg acattggccc tggacttaac ttgcttcaag
     1681 aggataaccc ggacttaatc gggagcgaaa ttgcccggtg gctttctacc ttagaaatca
     1741 gcggctaata tataactgtc tagaaataaa gagtatcatc tttcaaaggt cacccggcca
     1801 gcgacatgga ggcccagaat accctccttg acagtcttga cgtgcgcagc tcaggggcat
     1861 gatgtgactg tcgcccgtac atttagccca tacatcccca tgtataatca tttgcatcca
     1921 tacattttga tggccgcacg gcgcgaagca aaaattacgg ctcctcgctg cggacctgcg
     1981 agcagggaaa cgctcccctc acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga
     2041 gaaatataaa aggttaggat ttgccactga ggttcttctt tcatatactt ccttttaaaa
     2101 tcttgctagg atacagttct cacatcacat ccgaacataa acaaccatgg gtaaaaagcc
     2161 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     2221 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     2281 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     2341 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     2401 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     2461 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     2521 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     2581 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     2641 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     2701 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     2761 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     2821 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     2881 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     2941 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     3001 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     3061 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     3121 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aataatcagt
     3181 actgacaata aaaagattct tgttttcaag aacttgtcat ttgtatagtt tttttatatt
     3241 gtagttgttc tattttaatc aaatgttagc gtgatttata ttttttttcg cctcgacatc
     3301 atctgcccag atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga
     3361 atgctggtcg ctatactgct gtcgattcga tactaacgcc gccactagtt ctagagcggc
     3421 cgccaccgcg gtggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     3481 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3541 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     3601 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3661 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     3721 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3781 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3841 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3901 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3961 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4021 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4081 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4141 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4201 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4261 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4321 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4381 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4441 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4501 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4561 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4621 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4681 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4741 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4801 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4861 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4921 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4981 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5041 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5101 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5161 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5221 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5281 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5341 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5401 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5461 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5521 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5581 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
//
