LOCUS       sequence-254578-        6083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Complementation in Cryptococcus neoformans. Targets DNA constructs 
            to a Safe Haven Site 4, a small gene-free region. Contains the NEO 
            resistance marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6083)
  AUTHORS   Erpf PE, Stephenson CJ, Fraser JA
  TITLE     amdS as a dominant recyclable marker in Cryptococcus neoformans.
  JOURNAL   Fungal Genet Biol. 2019 Jun 17;131:103241. doi: 
            10.1016/j.fgb.2019.103241.
  PUBMED    31220607
REFERENCE   2  (bases 1 to 6083)
  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: "Fungal 
            Genet Biol."; date: "2019-06-17"; volume: "131:103241. doi"; pages: 
            " 10.1016/j.fgb.2019.103241"
COMMENT     Sequence Label: pPEE33
FEATURES             Location/Qualifiers
     source          1..6083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        794..898
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(899..967,968..1759)
                     /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(1117..1136)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1930..2518
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2419..2438
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2672..2689
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2806..2827
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2842..2847,2848..2865,2866..2872)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2880..2896
                     /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     2885..2907
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2904..2920
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2904..2920
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2916..3281
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     2939..2959
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2941..2959
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    2972..3079
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     2996..3012
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2996..3012
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3046..3062)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3047..3063)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3087..3106)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3088..3106)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3116..3133)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3116..3132)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3125..3147)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             4237..5037
                     /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="MSMIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQG
                     RPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEE
                     HQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
                     IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4297..4316)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4907..4926
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ttcgacacaa atgcatgtgt atctccacat ccatccctca ctaaccaacc caatgtcatc
       61 atgtagctgt gactctgcca tgtgcagtca tataataaaa agttacattg ttacatctcc
      121 ctgcgtttat aggggacaca ttgactgtct gctgaatgct atctttggtg atgtaaatac
      181 cagaaacccc catatccccg ccattgttta cttcatgtat gcatattacg tctaccctga
      241 agctgcaacg aatcccaatc caaacctatg aacatgaagg cgaaaatcat agtatgaatg
      301 ttgtggggta tatggcatag cacgatcaat ctgataaata ttaacacggg ccaaacggtt
      361 gcatcgcatt ctatctactt caactgctac gtctttggtg tgtttgttgc tctacagacg
      421 acccatgttt cccagtacac aggacctcct tcattatgtc accttttgtc ttcatccttg
      481 ccttcctccc atcctcgttc cttcctctcc tgcaaaaatt cctttttaaa agaacagcct
      541 gcgcgtttgg tggacttgta atccctccaa taaaacattc aacggcatag tcgtaaacaa
      601 gaatgttcac ctcctgattt cctcacattc cttcgccagc tctgtgaggt caaagagttc
      661 gtcaatagga agacgccgac caaaataaag acttgatgtg cggtaaggtg ctgggagggc
      721 caggcttggt acgaagggct ggaagagagt gccgctacag ggcgcgtcag gtggcacttt
      781 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      841 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
      901 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
      961 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     1021 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     1081 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     1141 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     1201 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     1261 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     1321 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     1381 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     1441 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     1501 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     1561 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     1621 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     1681 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     1741 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     1801 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     1861 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1921 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1981 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     2041 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     2101 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     2161 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     2221 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     2281 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     2341 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     2401 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2461 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     2521 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2581 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     2641 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     2701 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     2761 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     2821 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     2881 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc
     2941 aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg gccgctctag
     3001 aactagtgga tcccccgggc tgcaggaatt cgatatcaag cttatcgata ccgtcgacct
     3061 cgaggggggg cccggtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     3121 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     3181 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     3241 cccaacagtt gcgcagcctg aatggcgaat ggaaattgta agcgttaata ttttgttaaa
     3301 attcgcgtta aatttttgtt aaatcagctc atttttttaa ccaataggcc gaaatcggca
     3361 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtccatgg ctgcgaggat
     3421 gtgagctgga gagcggcgca cgggaagggg aggactcaca taagcatgca ggattcgagt
     3481 ggcatggtgt gcactgagtg tatggttgtc ggaggagagg atgatggtaa caacaatagc
     3541 agcaacgtca ctcgacgcgc gtccggtgtg ccacacgggg taacgccgag tcgccgtcag
     3601 ggtcgccgag accactctca cagcgtcacc gttggcacca gctcagctta cagcttctat
     3661 cctccgccag catccacata catcccctat accgcatccc ccacccactg cccaaggtga
     3721 gtcatcttcc cgcccccttc ccttgcccgc cactcagtcc tccatcctcc actaatccac
     3781 cttatcgcac ccaccgccta tcgcacatcc gagcacaatg ctgggcctgc caggggctgc
     3841 tagatggtgc tctccccacg ctgatctgca tgccggccat tggatcatgg gtgctaggtg
     3901 ctgggtgctg gatgttggat gctggatgct gggtgcacgc ttggtcattt ccttccagga
     3961 ttgacggtcg ccgagaggac gacgtggcgt tcgacaacga gggccgatag caccgcatcg
     4021 cctcgacctg catccatctc gccttgtcct tttggtgcaa caatccatcc gtgctggtgc
     4081 cacacgcata gctggaagag atggatgtgc gttgaacaga gctgccgtca ggactttttg
     4141 gtgcacggac cctattgtcc tccccaatct tcaccgcgtc tcctaatatg cagcctcttt
     4201 gctaattgtc tttttccatt agtaaactcg cccaacatgt ctatgattga acaagatgga
     4261 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     4321 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     4381 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     4441 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     4501 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatcccac
     4561 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     4621 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     4681 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     4741 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     4801 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4861 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4921 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4981 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagtg
     5041 aaggcggtaa ggggttaatt ttccttagag ggtgtatata tacatattag agaagtgata
     5101 caattttagc acactgcgaa ttcgagacag acatcgtgtc aatcatcttt tttgacatta
     5161 tatgcccatt aatctatcta cagacaacaa taccatcctt cccaccctca gcaacgccgt
     5221 tgaatcctca ggatcttcat ggctccttgt ctctgaaacc aggaagctag tttctacatc
     5281 tcttccgtgt taatacagat aaaccaaggg ggttaacgag agcatttagt ctagaaggga
     5341 acaagggact taccaatcag caacaatcac agctatccct ttctcctaat gccatttcaa
     5401 ggtatacttc tcatgcgcaa cgccgcaact cccattaact tctcttcctc aactgatccc
     5461 tcttctcctc actcatcttc aataacccgt ccgtaaaacc ggttcttaca atgaggatat
     5521 ctcctggctg aatgttgact tgtgcagtgt cattcttcgg cacatgcaat gagggtgcag
     5581 agtgaaggct cgttggctcg tgttaatcca cgcacgccgc aaggtaggca acgaaagccg
     5641 tagctgctat tactattttc atctagggcg cttcccttcg ataactcccg ttctcattct
     5701 tccctctctc tctaccttcc cagtactcac cgtctcctcc attcatgcaa ttcctgctgt
     5761 gaaaaccctc gctccgccct cacttcccta atggcccctt ccgaagtaag aaaagctatt
     5821 tccgactcct cactctcacc cgcatcatat cgagccctcc gcgaggatta gaaattccct
     5881 cgtgacgacc ccattgcata cggcggccac cttaaaaaca aagaccgacc atatcccctc
     5941 tctctccatc cctttcagta cgataccaac caccatatga ttcctttcat tagattgtcg
     6001 tctttcctcc ccgtcgcacg aattccttcc acgatttgac tgccgccacc gttaattaat
     6061 taacacaagt atcgtggcgc gcc
//
