LOCUS       Exported                6859 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Plasmid for tagging proteins in Cryptococcus neoformans; mCherry 
            tag, HYG selection.
ACCESSION   .
VERSION     .
KEYWORDS    YSBE 628 : pHYG_mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6859)
  AUTHORS   So YS, Yang DH, Jung KW, Huh WK, Bahn YS
  TITLE     Molecular Characterization of Adenylyl Cyclase Complex Proteins 
            Using Versatile Protein-Tagging Plasmid Systems in Cryptococcus 
            neoformans.
  JOURNAL   J Microbiol Biotechnol. 2017 Feb 28;27(2):357-364. doi: 
            10.4014/jmb.1609.09036.
  PUBMED    27780958
REFERENCE   2  (bases 1 to 6859)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6859
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(449..1474)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(2615..3322)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVRLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVPLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2893..2912)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3162..3180
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(3442..3459)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3442..3458)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3451..3473)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3596..3898
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3777..3796
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             4247..5041
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4301..4320)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4911..4930
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             5059..5919
                     /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"
     primer_bind     complement(5277..5296)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6090..6678
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6579..6598
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 gagccatgaa gatcctgagg attcaacggc gttgctgagg gtgggaagga tggtattgtt
      301 gtctgtagat agattaatgg gcatataatg tcaaaaaaga tgattgacac gatgtctgtc
      361 tcgaattcgc agtgtgctaa aattgtatca cttctctaat atgtatatat acaccctcta
      421 aggaaaatta accccttacc gccttcacct attcctttgc cctcggacga gtgctggggc
      481 gtcggtttcc actatcggcg agtacttcta cacagccatc ggtccagacg gccgcgcttc
      541 tgcgggcgat ttgtgtacgc ccgacagtcc cggctccgga tcggacgatt gcgtcgcatc
      601 gaccctgcgc ccaagctgca tcatcgaaat tgccgtcaac caagctctga tagagttggt
      661 caagaccaat gcggagcata tacgcccgga gccgcggcga tcctgcaagc tccggatgcc
      721 tccgctcgaa gtagcgcgtc tgctgctcca tacaagccaa ccacggcctc cagaagaaga
      781 tgttggcgac ctcgtattgg gaatccccga acatcgcctc gctccagtca atgaccgctg
      841 ttatgcggcc attgtccgtc aggacattgt tggagccgaa atccgcgtgc acgaggtgcc
      901 ggacttcggg gcagtcctcg gcccaaagca tcagctcatc gagagcctgc gcgacggacg
      961 cactgacggt gtcgtccatc acagtttgcc agtgatacac atggggatca gcaatcgcgc
     1021 atatgaaatc acgccatgta gtgtattgac cgattccttg cggtccgaat gggccgaacc
     1081 cgctcgtctg gctaagatcg gccgcagcga tcgcatccat ggcctccgcg accggctgca
     1141 gaacagcggg cagttcggtt tcaggcaggt cttgcaacgt gacaccctgt gcacggcggg
     1201 agatgcaata ggtcaggctc tcgctgaatt ccccaatgtc aagcacttcc ggaatcggga
     1261 gcgcggccga tgcaaagtgc cgataaacat aacgatcttt gtagaaacca tcggcgcagc
     1321 tatttacccg caggacatat ccacgccctc ctacatcgaa gctgaaagca cgagattctt
     1381 cgccctccga gagctgcatc aggtcggaga cgctgtcgaa cttttcgatc agaaacttct
     1441 cgacagacgt cgcggtgagt tcaggctttt tcatagacat gttgggcgag tttactaatg
     1501 gaaaaagaca attagcaaag aggctgcata ttaggagacg cggtgaagat tggggaggac
     1561 aatagggtcc gtgcaccaaa aagtcctgac ggcagctctg ttcaacgcac atccatctct
     1621 tccagctatg cgtgtggcac cagcacggat ggattgttgc accaaaagga caaggcgaga
     1681 tggatgcagg tcgaggcgat gcggtgctat cggccctcgt tgtcgaacgc cacgtcgtcc
     1741 tctcggcgac cgtcaatcct ggaaggaaat gaccaagcgt gcacccagca tccagcatcc
     1801 aacatccagc acccagcacc tagcacccat gatccaatgg ccggcatgca gatcagcgtg
     1861 gggagagcac catctagcag cccctggcag gcccagcatt gtgctcggat gtgcgatagg
     1921 cggtgggtgc gataaggtgg attagtggag gatggaggac tgagtggcgg gcaagggaag
     1981 ggggcgggaa gatgactcac cttgggcagt gggtggggga tgcggtatag gggatgtatg
     2041 tggatgctgg cggaggatag aagctgtaag ctgagctggt gccaacggtg acgctgtgag
     2101 agtggtctcg gcgaccctga cggcgactcg gcgttacccc gtgtggcaca ccggacgcgc
     2161 gtcgagtgac gttgctgcta ttgttgttac catcatcctc tcctccgaca accatacact
     2221 cagtgcacac catgccactc gaatcctgca tgcaagcttg gtaccgagct cggatccact
     2281 agtaacggcc gccagtgtgc tggaattcgc ccttgtacca atctatccct ctctccgaac
     2341 aatctccaca ttccctccct tctgctccct gaacccgcaa cacgagccgt ccacacttct
     2401 tctcaacttt gcgcttctca acttttgctt atgcaatctt gctccattat ctatcatccc
     2461 aacttatccc gactcttcct cgtcccaccg cccaccacac accacattac tttcaggctc
     2521 ctgaatacaa cacatcattc ataagttcaa gtatacgcca acatctcttc tttcgttata
     2581 aataaaaaga aagaaagaaa gaagaacgat tcttttactt gtacagctcg tccatgccgc
     2641 cggtggagtg gcggccctcg gcgcgttcgt actgttccac gatggtgtag tcctcgttgt
     2701 gggaggtgat gtccaacttg atgttgacgt tgtaggcgcc gggcagcggc acgggcttct
     2761 tggccttgta ggtggtcttg acctcagcgt cgtagtggcc gccgtccttc agcttcagcc
     2821 tctgcttgat ctcgcccttc agggcgccgt cctcggggta catccgctcg gaggaggcct
     2881 cccagcccat ggtcttcttc tgcattacgg ggccgtcgga ggggaagttg gtgccgcgca
     2941 gcctcacctt gtagatgaac tcgccgtcct gcagggagga gtcctgggtc acggtcacca
     3001 cgccgccgtc ctcgaagttc atcacgcgct cccacttgaa gccctcgggg aaggacagct
     3061 tcaagtagtc ggggatgtcg gcggggtgct tcacgtaggc cttggagccg tacatgaact
     3121 gaggggacag gatgtcccag gcgaagggca gggggccacc cttggtcacc ttcagcttgg
     3181 cggtctgggt gccctcgtag gggcggccct cgccctcgcc ctcgatctcg aactcgtggc
     3241 cgttcacgga gccctccatg tgcaccttga agcgcatgaa ctccttgatg atggccatgt
     3301 tatcctcctc gcccttgctc acagagccac cgccacctgc ggccgcaagg gcgaattctg
     3361 cagatatcca tcacactggc ggccgctcga gcatgcatct agagggccca attcgcccta
     3421 tagtgaatcg tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     3481 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     3541 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctatacg tacggcagtt
     3601 taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga
     3661 tattattgac acgccggggc gacggatggt gatccccctg gccagtgcac gtctgctgtc
     3721 agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat
     3781 gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct
     3841 cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat
     3901 gtcaggcatg agattatcaa aaaggatctt cacctagatc cttttcacgt agaaagccag
     3961 tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga
     4021 aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga
     4081 ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa
     4141 ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg
     4201 caggggatca agctctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     4261 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     4321 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     4381 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     4441 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4501 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4561 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4621 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4681 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4741 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     4801 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4861 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4921 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4981 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     5041 aattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5101 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5161 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5221 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5281 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5341 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5401 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5461 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5521 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5581 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5641 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5701 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5761 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5821 cgtatcgtag ttatctacac gacggggagc caggcaacta tggatgaacg aaatagacag
     5881 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     5941 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6001 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6061 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6121 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6181 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6241 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6301 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6361 ttggattcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6421 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6481 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6541 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6601 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6661 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6721 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     6781 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgaatca
     6841 gtgagcgagg aagcggaag
//
