LOCUS       sequence-120524-        6321 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Complementation in Cryptococcus neoformans. Targets DNA constructs 
            to a Safe Haven Site, a small gene-free region. Contains HYG 
            resistance marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6321)
  AUTHORS   Arras SD, Chitty JL, Blake KL, Schulz BL, Fraser JA
  TITLE     A genomic safe haven for mutant complementation in Cryptococcus 
            neoformans.
  JOURNAL   PLoS One. 2015 Apr 9;10(4):e0122916. doi: 
            10.1371/journal.pone.0122916. eCollection 2015.
  PUBMED    25856300
REFERENCE   2  (bases 1 to 6321)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2015-04-9"; volume: "10(4):e0122916. doi"; pages: " 
            10.1371/journal.pone.0122916. eCollection 2015"
COMMENT     Sequence Label: pSDMA58
FEATURES             Location/Qualifiers
     source          1..6321
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1824..2849)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(1824..2849)
                     /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(3811..4176)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     3945..3967
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3959..3976
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3960..3976
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3986..4005
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3986..4004
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    4013..4120
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     4029..4045
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4030..4046
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4080..4096)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4080..4096)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4133..4153)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4133..4151)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4172..4188)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4172..4188)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4185..4207)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4196..4212
                     /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(join(4220..4226,4227..4244,4245..4250))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4265..4286
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4403..4420)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4574..5162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4654..4673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5333..6124,6125..6193))
                     /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     5956..5975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6194..6298)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc aagatctgcc catagacttc aaatcatgga attggacgtt
       61 tatcttcttg ctttggatat ccaccctatc gtacaatctt ggcactttgg atcatacgag
      121 gcactttgcg gactagggaa tggttatcat caatttcttg gacctctcca tgacacctgg
      181 catcgtcgag ctaccacagc cccctgaatt cttaaaggcg aacccccctt cctaggaacc
      241 ttgagcgctg gactggatgg cactctgaga ccgcctatgc ttaaggacgc gagcttaccc
      301 ttccattgat ggaagtcgat catttatgat aatagaacac ggcggaactt gactttacag
      361 gccaagggaa gcaacaggag aagaagctaa ggttataaca ggctcagcat atttgtatat
      421 acggtgccat tcaacgctga acactttgta ctcacggtct gcccgttgaa taacaccatc
      481 tgctgatgat catcccaatt tgtgagtctt atttgccatt gatcctggtg tataaccgat
      541 actgaagccc tttaggtctg atcagttata gacgtggtat attcattctt cctagattag
      601 ctatgccgaa agagatggat accttgatgt gactggaatg ccatgaagca ggagattgta
      661 gcagctcaaa ttggagaagg atgttgcaac cgtcggccgt gaaggtcttg tcatatcttt
      721 gtctgcctgg gagaatatgt acaagtgccc cgatgttact gatggaaaac atcgtgtggc
      781 gcgccacgat acttgtgtta attaaaccaa accagtctgt gccaagtgga ttgcttatgc
      841 atcgagtggc gagcccactg acctctgggc cccttttttg aactataaaa agcggccaag
      901 tctgatttcg gtctcacaat catcaacatc gacttctctt ttcgcctcga tcaacctaaa
      961 caacggtcac tcagccatga actggacaat ggaatatttc gccttgcctt ggatctttgg
     1021 cctctagact caatcaacgt caggataaag gtgagcatag gttgctcact cactgtagcc
     1081 tttttttccc ttcttcttgg tccagatggt tcccaaggct ctataaagtg tatctgtcag
     1141 ttccacttcc ttactccgaa gcgattcaga gttgtaaagt gcgtctgacc ccctgattat
     1201 atctcaaagt cccttcacaa attcagacat gatgcgataa agtgccggat tgtcattcgc
     1261 gtcttgtgac tgtatagaaa gtgccggcat cctcttttct tttcctcttt ggccgagcga
     1321 ctcagagttg tgaagcctgc tggtatctgt ctgttccctt gccctgataa agaatatcga
     1381 agagcatttc tttgattcac aatacgttga cgaatggtgt gatggatcct aagcagcgcc
     1441 agccctttga acttgtgtcg aaaaaaaaaa aaaaacagca tatgatacgt aaagggaaga
     1501 aaaggatgga gctgactgtt ttgacaggca taattttgag cgtgcttcat tggccggttt
     1561 atctgtatta acacggaaga gatgtagaaa ctagcttcct ggtttcagag acaaggagcc
     1621 atgaagatcc tgaggattca acggcgttgc tgagggtggg aaggatggta ttgttgtctg
     1681 tagatagatt aatgggcata taatgtcaaa aaagatgatt gacacgatgt ctgtctcgaa
     1741 ttcgcagtgt gctaaaattg tatcacttct ctaatatgta tatatacacc ctctaaggaa
     1801 aattaacccc ttaccgcctt cacctattcc tttgccctcg gacgagtgct ggggcgtcgg
     1861 tttccactat cggcgagtac ttctacacag ccatcggtcc agacggccgc gcttctgcgg
     1921 gcgatttgtg tacgcccgac agtcccggct ccggatcgga cgattgcgtc gcatcgaccc
     1981 tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc tctgatagag ttggtcaaga
     2041 ccaatgcgga gcatatacgc ccggagccgc ggcgatcctg caagctccgg atgcctccgc
     2101 tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg gcctccagaa gaagatgttg
     2161 gcgacctcgt attgggaatc cccgaacatc gcctcgctcc agtcaatgac cgctgttatg
     2221 cggccattgt ccgtcaggac attgttggag ccgaaatccg cgtgcacgag gtgccggact
     2281 tcggggcagt cctcggccca aagcatcagc tcatcgagag cctgcgcgac ggacgcactg
     2341 acggtgtcgt ccatcacagt ttgccagtga tacacatggg gatcagcaat cgcgcatatg
     2401 aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc
     2461 gtctggctaa gatcggccgc agcgatcgca tccatggcct ccgcgaccgg ctgcagaaca
     2521 gcgggcagtt cggtttcagg caggtcttgc aacgtgacac cctgtgcacg gcgggagatg
     2581 caataggtca ggctctcgct gaattcccca atgtcaagca cttccggaat cgggagcgcg
     2641 gccgatgcaa agtgccgata aacataacga tctttgtaga aaccatcggc gcagctattt
     2701 acccgcagga catatccacg ccctcctaca tcgaagctga aagcacgaga ttcttcgccc
     2761 tccgagagct gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa cttctcgaca
     2821 gacgtcgcgg tgagttcagg ctttttcata gacatgttgg gcgagtttac taatggaaaa
     2881 agacaattag caaagaggct gcatattagg agacgcggtg aagattgggg aggacaatag
     2941 ggtccgtgca ccaaaaagtc ctgacggcag ctctgttcaa cgcacatcca tctcttccag
     3001 ctatgcgtgt ggcaccagca cggatggatt gttgcaccaa aaggacaagg cgagatggat
     3061 gcaggtcgag gcgatgcggt gctatcggcc ctcgttgtcg aacgccacgt cgtcctctcg
     3121 gcgaccgtca atcctggaag gaaatgacca agcgtgcacc cagcatccag catccaacat
     3181 ccagcaccca gcacctagca cccatgatcc aatggccggc atgcagatca gcgtggggag
     3241 agcaccatct agcagcccct ggcaggccca gcattgtgct cggatgtgcg ataggcggtg
     3301 ggtgcgataa ggtggattag tggaggatgg aggactgagt ggcgggcaag ggaagggggc
     3361 gggaagatga ctcaccttgg gcagtgggtg ggggatgcgg tataggggat gtatgtggat
     3421 gctggcggag gatagaagct gtaagctgag ctggtgccaa cggtgacgct gtgagagtgg
     3481 tctcggcgac cctgacggcg actcggcgtt accccgtgtg gcacaccgga cgcgcgtcga
     3541 gtgacgttgc tgctattgtt gttaccatca tcctctcctc cgacaaccat acactcagtg
     3601 cacaccatgc cactcgaatc ctgcatgctt atgtgagtcc tccccttccc gtgcgccgct
     3661 ctccagctca catcctcgca gccatggact caaccctatc tcggtctatt cttttgattt
     3721 ataagggatt ttgccgattt cggcctattg gttaaaaaaa tgagctgatt taacaaaaat
     3781 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
     3841 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     3901 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     3961 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
     4021 gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc agcccggggg
     4081 atccactagt tctagagcgg ccgccaccgc ggtggagctc cagcttttgt tccctttagt
     4141 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4201 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4261 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4321 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4381 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4441 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4501 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4561 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4621 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4681 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4741 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4801 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4861 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4921 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4981 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     5041 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5101 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5161 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5221 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5281 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5341 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5401 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5461 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5521 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5581 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5641 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5701 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5761 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5821 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5881 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5941 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6001 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6061 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6121 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6181 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6241 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6301 catttccccg aaaagtgcca c
//
