LOCUS       sequence-370817-        7709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid for in vivo gene amplification (HamAmp) at RPL25 locus 
            (~11 copy).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7709)
  TITLE     Vickers Lab plasmids
REFERENCE   2  (bases 1 to 7709)
  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"
COMMENT     Sequence Label: pILGFP3AE4
FEATURES             Location/Qualifiers
     source          1..7709
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(31..47)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(31..47)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(44..66)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    55..71
                     /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(79..85,86..103,104..109))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    124..145
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(262..279)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(433..1021)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(513..532)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1192..1983,1984..2052))
                     /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     1815..1834
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2053..2157)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2225..2243
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2281..2303)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2403..2422
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2616..2638
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2630..2647
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2631..2647
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5319..5352)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5563..5976
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             5999..6709
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
ORIGIN
        1 catagtttaa acaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
       61 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
      121 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
      181 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
      241 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
      301 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
      361 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
      421 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
      481 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
      541 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
      601 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
      661 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
      721 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
      781 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
      841 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
      901 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
      961 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     1021 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     1081 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     1141 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     1201 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     1261 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     1321 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     1381 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     1441 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     1501 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     1561 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     1621 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     1681 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     1741 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     1801 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     1861 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     1921 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     1981 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     2041 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     2101 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     2161 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     2221 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     2281 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     2341 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     2401 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     2461 atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg
     2521 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     2581 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     2641 ggccagtgaa ttcagtttaa acatgtacta atcagtctaa caccccggag gaggcttatc
     2701 attacatatt cttgtctcgg cgttctttaa gtttcttatc ttgtatgccc gatatagcaa
     2761 ccttgttggt accaatctaa cggtttccgt actttgcaat gaagagatga ggaggcatgg
     2821 gtcacttatt taatatgtac gggtgtttac atggagttgc tttctttttt tgtctcagca
     2881 gtcattgtgc gccaaaaaaa gagaaaaccg tgagccgaag tccacgctct ggagttaggc
     2941 tctcccatta cggagagaag catttcctca gcctgggagc cccgttggaa cagtcaggct
     3001 aaactgggcc ttcctaccca ctgcttgctg tttctcactg gacgcacaag gggattttct
     3061 ttctaccttc ggcttgcctc actgcgttgg ggcttcccaa tgcaacttcg ttcgtatgca
     3121 tacaatcttt tagatattat cttttaaaat tattttaaaa caattttaaa tgtatctcat
     3181 atgcttttct tctgctgttg aaaaggctaa acaaagaaga tcaataagat aaagcggccg
     3241 caaaatgtct aagaatatcg ttgtcctacc gggtgatcac gtcggtaaag aagttactga
     3301 cgaagctatt aaggtcttga atgccattgc tgaagtccgt ccagaaatta agttcaattt
     3361 ccaacatcac ttgatcgggg gtgctgccat cgatgccact ggcactcctt taccagatga
     3421 agctctagaa gcctctaaga aagccgatgc tgtcttacta ggtgctgttg gtggtccaaa
     3481 atggggtacg ggcgcagtta gaccagaaca aggtctattg aagatcagaa aggaattggg
     3541 tctatacgcc aacttgagac catgtaactt tgcttctgat tctttactag atctttctcc
     3601 tttgaagcct gaatatgcaa agggtaccga tttcgtcgtc gttagagaat tggttggtgg
     3661 tatctacttt ggtgaaagaa aagaagatga aggtgacgga gttgcttggg actctgagaa
     3721 atacagtgtt cctgaagttc aaagaattac aagaatggct gctttcttgg cattgcaaca
     3781 aaacccacca ttaccaatct ggtctcttga caaggctaac gtgcttgcct cttccagatt
     3841 gtggagaaag actgttgaag aaaccatcaa gactgagttc ccacaattaa ctgttcagca
     3901 ccaattgatc gactctgctg ctatgatttt ggttaaatca ccaactaagc taaacggtgt
     3961 tgttattacc aacaacatgt ttggtgatat tatctccgat gaagcctctg ttattccagg
     4021 ttctttgggt ttattacctt ctgcatctct agcttcccta cctgacacta acaaggcatt
     4081 cggtttgtac gaaccatgtc atggttctgc cccagattta ccagcaaaca aggttaaccc
     4141 aattgctacc atcttatctg cagctatgat gttgaagtta tccttggatt tggttgaaga
     4201 aggtagggct cttgaagaag ctgttagaaa tgtcttggat gcaggtgtca gaaccggtga
     4261 ccttggtggt tctaactcta ccactgaggt tggcgatgct atcgccaagg ctgtcaagga
     4321 aatcttggct taaagagtct tttgtaacga ccccgtctcc accaacttgg tatgcttgaa
     4381 atctcaaggc cattacacat tcagttatgt gaacgaaagg tctttattta acgtagcata
     4441 aactaaataa tacaggttcc ggttagcctg cgggcccaaa gctagctcta attggtttaa
     4501 ttaataaatt taatattatt tttaaatttt tctttaaata tacaataaat ctttcataac
     4561 atgttaaatt catgattaag cgtaaataaa gtgtagtggc agagtgcacg gggtttcctg
     4621 tgccttacaa agtaggtacc aatttgcgta ttgcagcgag ggttccggtt actatttata
     4681 attacgtgtt agtgtactgt gattttattg aggctataac aagaaaagga tctgtgaagg
     4741 ttttgagaga atgaggcgaa aacacagggt tattttcatc agcaacgccg ctgtttcagc
     4801 ggcaccctgc ccttttcttt tcgcaagatg agagtaaaga gttgtacatc aggtaagaat
     4861 gttattattt aaattcgaag tgataaattc ttttcatgat gaatcactcg cttatatggg
     4921 gtagaatata tatatatgtg tgtgtgtgtg tgtgtttgtg tatgtaggga tggtgcgcgt
     4981 ttgttgtgtg acatttgcta ctcattcttt tccttttcct acgactggct taacgggaat
     5041 attatcaatt tgctgcattc ttatgcttcg gtccgatgct caagcttaac ttcttcgtga
     5101 ggaaggaaag tgttgtctcc tacttttttc aaattttcga attgtattta tatttattta
     5161 gtacttcttg agtttacata tccttcgtaa aaatgcaact tttgtcgaaa aacacttcca
     5221 aaaaaaaata ataatgaatt tatgaagcat actaacgagc gagcacatcg ctgaggtatc
     5281 attacttcat gagataaatt aactcgagct cgagcctaat aacttcgtat agcatacatt
     5341 atacgaagtt atattaaggg ttgctcgacc agaaagcgac cacccaactt tggctgataa
     5401 tagcgtataa acaatgcata ctttgtacgt tcaaaataca atgcagtaga tatatttatg
     5461 catattacat ataatacata tcacatagga agcaacaggc gcgttggact tttaattttc
     5521 gaggaccgcg aatccttaca tcacacccaa tcccccacaa gtgatccccc acacaccata
     5581 gcttcaaaat gtttctactc cttttttact cttccagatt ttctcggact ccgcgcatcg
     5641 ccgtaccact tcaaaacacc caagcacagc atactaaatt tcccctcttt cttcctctag
     5701 ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa aaagagaccg cctcgtttct
     5761 ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt ttctttttct tgaaaatttt
     5821 tttttttgat ttttttctct ttcgatgacc tcccattgat atttaagtta ataaacggtc
     5881 ttcaatttct caagtttcag tttcattttt cttgttctat tacaactttt tttacttctt
     5941 gctcattaga aagaaagcat agcaatctaa tctaagtttt aattacaaaa tgggatcctc
     6001 taaaggtgaa gaattattca ctggtgttgt cccaattttg gttgaattag atggtgatgt
     6061 taatggtcac aaattttctg tctccggtga aggtgaaggt gatgctactt acggtaaatt
     6121 gaccttaaaa tttatttgta ctactggtaa attgccagtt ccatggccaa ccttagtcac
     6181 tactttcggt tatggtgttc aatgttttgc gagataccca gatcatatga aacaacatga
     6241 ctttttcaag tctgccatgc cagaaggtta tgttcaagaa agaactattt ttttcaaaga
     6301 tgacggtaac tacaagacca gagctgaagt caagtttgaa ggtgatacct tagttaatag
     6361 aatcgaatta aaaggtattg attttaaaga agatggtaac attttaggtc acaaattgga
     6421 atacaactat aactctcaca atgtttacat catggctgac aaacaaaaga atggtatcaa
     6481 agttaacttc aaaattagac acaacattga agatggttct gttcaattag ctgaccatta
     6541 tcaacaaaat actccaattg gtgatggtcc agtcttgtta ccagacaacc attacttatc
     6601 cactcaatct gccttatcca aagatccaaa cgaaaagaga gaccacatgg tcttgttaga
     6661 atttgttact gctgctggta ttacccatgg tatggatgaa ttgtacaaaa gatcttaaac
     6721 tagtaggact atttgcaaag ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag
     6781 caggctggga agcatatttg agaagatgcg gccagcaaaa ctaaaaaact gtattataag
     6841 taaatgcatg tatactaaac tcacaaatta gagcttcaat ttaattatat cagttattac
     6901 ccgggaatct cggtcgtaat gatttgcatg ctgcgatact gccgtagcgg gccttcgtat
     6961 agctcggccg agctcgtaca aaaggcaagc agtgtatcgg acagagctga tataacacaa
     7021 tacgctcgta gtcgatgcat gccgtggctg ctctcggtcg ggtataagtc ttagacaata
     7081 gtcttacctc gcatgtataa taaatctttt gtatttaatc tattatatgt ttctatgctt
     7141 ttttttccta ttgttgtttg cttttccttt tccttatttc tttctagctt ctaattttct
     7201 ttcttttttt tttttttttc attgaaaatt atatatatat atatatatca gaacaattgt
     7261 ccagtattga acaatacagg ttatttcgaa caattgaaaa aaaaaaatca cagaaaaaca
     7321 tatcgagaaa agggtcatgg ctccatctgg tatgtgaact gcaatattaa tagcacgaga
     7381 aaattgagag gaagatagat gggaactagt agagttgata ttgatgagat acgaaaacca
     7441 cacgtaaata aactatccga cgacaagaat agtgcattaa ggacttgatt aagatatggt
     7501 gagcaacgta attatcgggc tcaacagttt attagcaatc gttttgatag aagcgttgat
     7561 gctgtggaag ttgttctttt tactaccaac agccattaac aaatccatta gagtgttcgt
     7621 tcgtttttct gctcagattc aagaaaatta ttccatcctc attatacttt tttcttctat
     7681 ttcgtgctcc acgtcgaggt atcaaggaa
//
