LOCUS       Exported                6271 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Plasmid for tagging proteins in Cryptococcus neoformans; 4xFLAG tag,
            HYG selection.
ACCESSION   .
VERSION     .
KEYWORDS    YSBE 731 : pHYG_4xFLAG
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6271)
  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 6271)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6271
                     /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             1019..2044
                     /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(2624..2647)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(2657..2680)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(2684..2707)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(2711..2734)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2854..2871)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2854..2870)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2863..2885)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3008..3310
                     /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     3189..3208
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3659..4453
                     /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(3713..3732)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4323..4342
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             4471..5331
                     /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(4689..4708)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5502..6090
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5991..6010
                     /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 catgcaggat tcgagtggca tggtgtgcac tgagtgtatg gttgtcggag gagaggatga
      301 tggtaacaac aatagcagca acgtcactcg acgcgcgtcc ggtgtgccac acggggtaac
      361 gccgagtcgc cgtcagggtc gccgagacca ctctcacagc gtcaccgttg gcaccagctc
      421 agcttacagc ttctatcctc cgccagcatc cacatacatc ccctataccg catcccccac
      481 ccactgccca aggtgagtca tcttcccgcc cccttccctt gcccgccact cagtcctcca
      541 tcctccacta atccacctta tcgcacccac cgcctatcgc acatccgagc acaatgctgg
      601 gcctgccagg ggctgctaga tggtgctctc cccacgctga tctgcatgcc ggccattgga
      661 tcatgggtgc taggtgctgg gtgctggatg ttggatgctg gatgctgggt gcacgcttgg
      721 tcatttcctt ccaggattga cggtcgccga gaggacgacg tggcgttcga caacgagggc
      781 cgatagcacc gcatcgcctc gacctgcatc catctcgcct tgtccttttg gtgcaacaat
      841 ccatccgtgc tggtgccaca cgcatagctg gaagagatgg atgtgcgttg aacagagctg
      901 ccgtcaggac tttttggtgc acggacccta ttgtcctccc caatcttcac cgcgtctcct
      961 aatatgcagc ctctttgcta attgtctttt tccattagta aactcgccca acatgtctat
     1021 gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
     1081 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
     1141 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
     1201 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
     1261 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
     1321 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc
     1381 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
     1441 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
     1501 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
     1561 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
     1621 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
     1681 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
     1741 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
     1801 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
     1861 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
     1921 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
     1981 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
     2041 ataggtgaag gcggtaaggg gttaattttc cttagagggt gtatatatac atattagaga
     2101 agtgatacaa ttttagcaca ctgcgaattc gagacagaca tcgtgtcaat catctttttt
     2161 gacattatat gcccattaat ctatctacag acaacaatac catccttccc accctcagca
     2221 acgccgttga atcctcagga tcttcatggc tccaagcttg gtaccgagct cggatccact
     2281 agtaacggcc gccagtgtgc tggaattcgc ccttgtacca atctatccct ctctccgaac
     2341 aatctccaca ttccctccct tctgctccct gaacccgcaa cacgagccgt ccacacttct
     2401 tctcaacttt gcgcttctca acttttgctt atgcaatctt gctccaccat ctatcatccc
     2461 aacttatccc gactcttcct cgtcccaccg cccaccacac accacattac tttcaggctc
     2521 ctgaatacaa cacatcattc ataagttcaa gtatacgcca acatctcttc tttcgttata
     2581 aataaaaaga aagaaagaaa gaagaacgat tcttttagtc gaccttatcg tcgtcatcct
     2641 tgtaatcttc tagacacttg tcgtcgtcgt ccttgtagtc tcccttatcg tcatcgtctt
     2701 tgtagtcacc cttatcgtcg tcatccttgt aatctggcgc gccgcatgct gcggccgcaa
     2761 gggcgaattc tgcagatatc catcacactg gcggccgctc gagcatgcat ctagagggcc
     2821 caattcgccc tatagtgaat cgtattacaa ttcactggcc gtcgttttac aacgtcgtga
     2881 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     2941 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctata
     3001 cgtacggcag tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga
     3061 tgtacagagt gatattattg acacgccggg gcgacggatg gtgatccccc tggccagtgc
     3121 acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga
     3181 aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga
     3241 agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg
     3301 gggaatataa atgtcaggca tgagattatc aaaaaggatc ttcacctaga tccttttcac
     3361 gtagaaagcc agtccgcaga aacggtgctg accccggatg aatgtcagct actgggctat
     3421 ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg
     3481 gcgatagcta gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc
     3541 gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag
     3601 gatctgatgg cgcaggggat caagctctga tcaagagaca ggatgaggat cgtttcgcat
     3661 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3721 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3781 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3841 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3901 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3961 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     4021 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     4081 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     4141 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg
     4201 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     4261 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     4321 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     4381 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     4441 cgagttcttc tgaattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4501 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4561 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4621 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4681 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4741 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4801 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4861 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4921 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4981 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     5041 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5101 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5161 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5221 ggtaagccct cccgtatcgt agttatctac acgacgggga gccaggcaac tatggatgaa
     5281 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5341 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5401 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5461 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5521 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5581 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5641 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5701 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5761 tgtcttaccg ggttggattc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5821 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5881 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5941 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6001 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6061 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6121 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     6181 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     6241 cgcagcgaat cagtgagcga ggaagcggaa g
//
