LOCUS       pBS-SKII-3XHA-eD        4770 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 4770)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2022 from SnapGene 6.1.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4770
                     /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..2508
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        1389..1732
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1733..2305
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      2311..2508
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        complement(2582..2600)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2621..2637)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2645..2661
                     /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(2669..2699)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2714..2735
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3023..3611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3782..4642)
                     /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(4643..4747)
                     /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 ccgttgccag
     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 ccatgggtac
     1741 cactcttgac gacacggctt accggtaccg caccagtgtc ccgggggacg ccgaggccat
     1801 cgaggcactg gatgggtcct tcaccaccga caccgtcttc cgcgtcaccg ccaccgggga
     1861 cggcttcacc ctgcgggagg tgccggtgga cccgcccctg accaaggtgt tccccgacga
     1921 cgaatcggac gacgaatcgg acgacgggga ggacggcgac ccggactccc ggacgttcgt
     1981 cgcgtacggg gacgacggcg acctggcggg cttcgtggtc gtctcgtact ccggctggaa
     2041 ccgccggctg accgtcgagg acatcgaggt cgccccggag caccgggggc acggggtcgg
     2101 gcgcgcgttg atggggctcg cgacggagtt cgcccgcgag cggggcgccg ggcacctctg
     2161 gctggaggtc accaacgtca acgcaccggc gatccacgcg taccggcgga tggggttcac
     2221 cctctgcggc ctggacaccg ccctgtacga cggcaccgcc tcggacggcg agcaggcgct
     2281 ctacatgagc atgccctgcc cctaatcagt actgacaata aaaagattct tgttttcaag
     2341 aacttgtcat ttgtatagtt tttttatatt gtagttgttc tattttaatc aaatgttagc
     2401 gtgatttata ttttttttcg cctcgacatc atctgcccag atgcgaagtt aagtgcgcag
     2461 aaagtaatat catgcgtcaa tcgtatgtga atgctggtcg ctatactgct gtcgattcga
     2521 tactaacgcc gccactagtt ctagagcggc cgccaccgcg gtggagctcc agcttttgtt
     2581 ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt
     2641 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     2701 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     2761 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     2821 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     2881 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     2941 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3001 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3061 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3121 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3181 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3241 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3301 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3361 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3421 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     3481 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3541 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3601 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3661 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     3721 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     3781 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     3841 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     3901 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     3961 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4021 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4081 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4141 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4201 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4261 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4321 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4381 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4441 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4501 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4561 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     4621 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     4681 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     4741 ttccgcgcac atttccccga aaagtgccac
//
