LOCUS       sequence-120525-        5862 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 
            nourseothricin resistance marker (NAT).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5862)
  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 5862)
  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: pSDMA25
FEATURES             Location/Qualifiers
     source          1..5862
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1815..2378)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="TLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFTLR
                     EVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRRLT
                     VEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTLCG
                     LDTALYDGTASDGEQALYMSMPCP"
     CDS             complement(3352..3717)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     3486..3508
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3500..3517
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3501..3517
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3527..3546
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3527..3545
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    3554..3661
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     3570..3586
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3571..3587
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3621..3637)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3621..3637)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3674..3694)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3674..3692)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3713..3729)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3713..3729)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3726..3748)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3737..3753
                     /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(3761..3767,3768..3785,3786..3791))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3806..3827
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3944..3961)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4115..4703)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4195..4214)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4874..5665,5666..5734))
                     /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     5497..5516
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5735..5839)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctcatcagat ctgcccatag acttcaaatc atggaattgg acgtttatct tcttgctttg
       61 gatatccacc ctatcgtaca atcttggcac tttggatcat acgaggcact ttgcggacta
      121 gggaatggtt atcatcaatt tcttggacct ctccatgaca cctggcatcg tcgagctacc
      181 acagccccct gaattcttaa aggcgaaccc cccttcctag gaaccttgag cgctggactg
      241 gatggcactc tgagaccgcc tatgcttaag gacgcgagct tacccttcca ttgatggaag
      301 tcgatcattt atgataatag aacacggcgg aacttgactt tacaggccaa gggaagcaac
      361 aggagaagaa gctaaggtta taacaggctc agcatatttg tatatacggt gccattcaac
      421 gctgaacact ttgtactcac ggtctgcccg ttgaataaca ccatctgctg atgatcatcc
      481 caatttgtga gtcttatttg ccattgatcc tggtgtataa ccgatactga agccctttag
      541 gtctgatcag ttatagacgt ggtatattca ttcttcctag attagctatg ccgaaagaga
      601 tggatacctt gatgtgactg gaatgccatg aagcaggaga ttgtagcagc tcaaattgga
      661 gaaggatgtt gcaaccgtcg gccgtgaagg tcttgtcata tctttgtctg cctgggagaa
      721 tatgtacaag tgccccgatg ttactgatgg aaaacatcgt gtggcgcgcc acgatacttg
      781 tgttaattaa accaaaccag tctgtgccaa gtggattgct tatgcatcga gtggcgagcc
      841 cactgacctc tgggcccctt ttttgaacta taaaaagcgg ccaagtctga tttcggtctc
      901 acaatcatca acatcgactt ctcttttcgc ctcgatcaac ctaaacaacg gtcactcagc
      961 catgaactgg acaatggaat atttcgcctt gccttggatc tttggcctct agactcaatc
     1021 aacgtcagga taaaggtgag cataggttgc tcactcactg tagccttttt ttcccttctt
     1081 cttggtccag atggttccca aggctctata aagtgtatct gtcagttcca cttccttact
     1141 ccgaagcgat tcagagttgt aaagtgcgtc tgaccccctg attatatctc aaagtccctt
     1201 cacaaattca gacatgatgc gataaagtgc cggattgtca ttcgcgtctt gtgactgtat
     1261 agaaagtgcc ggcatcctct tttcttttcc tctttggccg agcgactcag agttgtgaag
     1321 cctgctggta tctgtctgtt cccttgccct gataaagaat atcgaagagc atttctttga
     1381 ttcacaatac gttgacgaat ggtgtgatgg atcctaagca gcgccagccc tttgaacttg
     1441 tgtcgaaaaa aaaaaaaaaa cagcatatga tacgtaaagg gaagaaaagg atggagctga
     1501 ctgttttgac aggcataatt ttgagcgtgc ttcattggcc ggtttatctg tattaacacg
     1561 gaagagatgt agaaactagc ttcctggttt cagagacaag gagccatgaa gatcctgagg
     1621 attcaacggc gttgctgagg gtgggaagga tggtattgtt gtctgtagat agattaatgg
     1681 gcatataatg tcaaaaaaga tgattgacac gatgtctgtc tcgaattcgc agtgtgctaa
     1741 aattgtatca cttctctaat atgtatatat acaccctcta aggaaaatta accccttacc
     1801 gccttcacga attcttaggg gcagggcatg ctcatgtaga gcgcctgctc gccgtccgag
     1861 gcggtgccgt cgtacagggc ggtgtccagg ccgcagaggg tgaaccccat ccgccggtac
     1921 gcgtggatag ccggtgcgtt aacgttggtg acctccagcc agaggtgccc ggcgccccgc
     1981 tcgcgggcga actccgtcgc gagccccatc aacgcgcgcc cgaccccgtg cccccggtgc
     2041 tccggggcga cctcgatgtc ctcgacggtc agccggcggt tccagccgga gtacgagacg
     2101 accacgaagc ccgccaggtc gccgtcgtcc ccgtacgcga cgaacgtccg ggagtccggg
     2161 tcgccgtcct ccccgtcgtc cgattcgtcg tccgattcgt cgtcggggaa caccttggtc
     2221 aggggcgggt ccaccggcac ctcccgcagg gtgaagccgt ccccggtggc ggtgacgcgg
     2281 aagacggtgt cggtggtgaa ggacccatcc agtgcctcga tggcctcggc gtcccccggg
     2341 acactggtgc ggtaccggta agccgtgtcg tcaagagtgg cggccgccat agacatgttg
     2401 ggcgagttta ctaatggaaa aagacaatta gcaaagaggc tgcatattag gagacgcggt
     2461 gaagattggg gaggacaata gggtccgtgc accaaaaagt cctgacggca gctctgttca
     2521 acgcacatcc atctcttcca gctatgcgtg tggcaccagc acggatggat tgttgcacca
     2581 aaaggacaag gcgagatgga tgcaggtcga ggcgatgcgg tgctatcggc cctcgttgtc
     2641 gaacgccacg tcgtcctctc ggcgaccgtc aatcctggaa ggaaatgacc aagcgtgcac
     2701 ccagcatcca gcatccaaca tccagcaccc agcacctagc acccatgatc caatggccgg
     2761 catgcagatc agcgtgggga gagcaccatc tagcagcccc tggcaggccc agcattgtgc
     2821 tcggatgtgc gataggcggt gggtgcgata aggtggatta gtggaggatg gaggactgag
     2881 tggcgggcaa gggaaggggg cgggaagatg actcaccttg ggcagtgggt gggggatgcg
     2941 gtatagggga tgtatgtgga tgctggcgga ggatagaagc tgtaagctga gctggtgcca
     3001 acggtgacgc tgtgagagtg gtctcggcga ccctgacggc gactcggcgt taccccgtgt
     3061 ggcacaccgg acgcgcgtcg agtgacgttg ctgctattgt tgttaccatc atcctctcct
     3121 ccgacaacca tacactcagt gcacaccatg ccactcgaat cctgcatgct tatgtgagtc
     3181 ctccccttcc cgtgcgccgc tctccagctc acatcctcgc agccatggac tcaaccctat
     3241 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3301 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3361 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     3421 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     3481 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
     3541 atagggcgaa ttgggtaccg ggccccccct cgaggtcgac ggtatcgata agcttgatat
     3601 cgaattcctg cagcccgggg gatccactag ttctagagcg gccgccaccg cggtggagct
     3661 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
     3721 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3781 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3841 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3901 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     3961 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4021 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4081 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4141 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4201 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4261 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4321 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4381 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4441 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4501 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     4561 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4621 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4681 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4741 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4801 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4861 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4921 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4981 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5041 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5101 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5161 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5221 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5281 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5341 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5401 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5461 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5521 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5581 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5641 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5701 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5761 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5821 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc ac
//
