LOCUS       sequence-370824-        8191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid for in vivo gene amplification (HamAmp) at SEC23 locus (4 
            copy).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8191)
  TITLE     Vickers Lab plasmids
REFERENCE   2  (bases 1 to 8191)
  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: pILGFP5EA4
FEATURES             Location/Qualifiers
     source          1..8191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1095..1111)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1095..1111)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1108..1130)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1119..1135
                     /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(1143..1149,1150..1167,1168..1173))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1188..1209
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1326..1343)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1497..2085)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1577..1596)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2256..3047,3048..3116))
                     /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     2879..2898
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3117..3221)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3289..3307
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3345..3367)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3467..3486
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3680..3702
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3694..3711
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3695..3711
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4668..5696
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             4668..5696
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      5702..5899
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    complement(6646..6679)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        6890..7303
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             7326..8036
                     /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 aaattagagc ttcaatttaa ttatatcagt tattacccgg gaatctcggt cgtaatgatt
       61 tgatatgaag gcatgcagtt cattgatgtt gaagaagtgg actccaccgg atattgaatc
      121 acatgggatt tcagatttca agtccaaatt ttcgattggg aatttttggt caaggctttt
      181 ttctacgcgg gctattgcag gtgagcaaga catagaatca caggccggac taatgtcaaa
      241 cgaagaagtc ttgtaaataa caggtaagtc taatttattt aaaaaaatcc acgagcatat
      301 agatttataa caataataat aattaatatt catcaatttt ttagtaaatt catatatttt
      361 actcctactc tatgtatttt cagtaagtct actagtcgta ttacccgtcg gtttcaaggg
      421 atattatgga ggtaataagg atttttttac cgatttaaaa acccattata tccctcttag
      481 aaaaatttta gtattcttag ataagttgct agatttacca atatcaaagg gaagactgca
      541 ggacaacaca aaaacacaag aaaaggagga caaaaaatgg acttcgagac taatgaagac
      601 atcaacgggg tccgtttcac ctggaacgtg tttccctcta ctagaagcga cgctaacagc
      661 aacgtcgtcc ctgtgggctg tttgtacaca cctttgaagg agtatgacga attgaacgtt
      721 gccccataca atccagttgt gtgttccggt ccacactgta agtcgattct aaatccttac
      781 tgtgtgattg atccgcgcaa ttcgtcctgg agttgtccca tctgtaactc caggaaccat
      841 ttacctccac agtacaccaa cttgtcacag gagaatatgc cgctagagtt acagtccacc
      901 accattgagt acatcaccaa taagcccgtt accgttcctc ccattttctt ctttgttgtg
      961 gatttgactt cggaaaccga gaacctcgat tcgctaaagg agtctatcat cacttctttg
     1021 tctctattac ctcccaacgc gctgattggg ttgatcactt acggaaacgt ttaaacaagc
     1081 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     1141 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     1201 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1261 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     1321 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1381 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1441 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1501 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1561 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     1621 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     1681 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     1741 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     1801 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     1861 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     1921 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     1981 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2041 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2101 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2161 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2221 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2281 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     2341 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     2401 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     2461 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     2521 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     2581 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     2641 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     2701 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     2761 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     2821 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     2881 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     2941 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3001 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3061 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3121 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3181 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3241 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     3301 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     3361 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     3421 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     3481 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     3541 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     3601 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     3661 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattcagt
     3721 ttaaactctt ctgcttcgtt cagctgcagg ttaaagtagt tgtccgtgga cacgagcgta
     3781 cctctatatt cggtgctgtt gaacttaagc ttgacgccta cacgatggtt gaccaggcct
     3841 ttgaggaacg gcttcgggtt caccggctgc atcgcgctga tatcactgct ctcgctcatt
     3901 gtcttgttgt gttgtgacgt agtgtgtggt gtggtatagt acgcggtgct gtgtgtgctg
     3961 tgctgttttt gcgttgctca tagaaccgtt gtttcttgct cttaccatct catgttcata
     4021 tatgttgggg aaaagaggtg gaacaagaaa agttacccgg acggcagagc ggttgttttg
     4081 cctgctttgc cataacacag gatgggatca cacacccccc aagccatgct acaacttgga
     4141 agagtgcaat tcaagttggt tgtcctcagt agtttgagag attgcgctaa ttttgtttta
     4201 tcaccgttat ttactgtcag ttgagatttg tcgcttgcga cgactagccc gccgcctctc
     4261 gtttgttaca aaggaggaaa gttctagcaa gaataaaaga aaaacacgtt tgaaaaaaga
     4321 aaaaaagata cgggcccgaa taccctcctt gacagtcttg acgtgcgcag ctcaggggca
     4381 tgatgtgact gtcgcccgta catttagccc atacatcccc atgtataatc atttgcatcc
     4441 atacattttg atggccgcac ggcgcgaagc aaaaattacg gctcctcgct gcggacctgc
     4501 gagcagggaa acgctcccct cacagacgcg ttgaattgtc cccacgccgc gcccctgtag
     4561 agaaatataa aaggttagga tttgccactg aggttcttct ttcatatact tccttttaaa
     4621 atcttgctag gatacagttc tcacatcaca tccgaacata aacaaccatg ggtaaaaagc
     4681 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
     4741 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
     4801 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
     4861 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
     4921 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
     4981 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
     5041 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
     5101 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
     5161 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
     5221 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
     5281 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     5341 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     5401 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
     5461 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     5521 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     5581 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     5641 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaataatcag
     5701 tactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat
     5761 tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat
     5821 catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg
     5881 aatgctggtc gctatactgc tgtcgattcg atactaacgg cggccgcgag caacggcttt
     5941 cttttgtttt atttgatcgt tattaggctt tttgtataat ataaggcctg ttcagtccat
     6001 tgcgcgtgca agaattttcc tttggacctg aacgaaaaga gtagaggatt ttatataata
     6061 tattctcctt cttttttgta ctgcgtggcc tttactacga atatctgtat ctaatttctt
     6121 ttctaacttt tttattacca ttactactac tactattatt attattatta ttattattat
     6181 tattgtgtat gactataatg ctttactgcc tccaattatg ctttttttat tatttttttt
     6241 ggctgaaact acagtatagt aggtatgtat atatctaaaa ctcaaaaaga ggcatgtcta
     6301 atgtaatacc atcaaaaaca atgaaattat ttaatggaga tagcctttta cctagaatgt
     6361 taatgacatc ctgcgctaca gcacccccta taatggccgc gacaggagca aactcgatac
     6421 ctttctgttt gataaactgt tgtatgtaat cgtcctttac cactgttgct ggtacaccca
     6481 gattttcaca ccatacggcc gcatctcttt tcatttgtat ttaaatccat ttcaaatttt
     6541 atgtttagtt cgagatcctc agttttcggc gcataggaac cacgtacata ataactaaac
     6601 ataaatctat aataaataaa aaacaacgat gggagctcga gcctaataac ttcgtatagc
     6661 atacattata cgaagttata ttaagggttg ctcgaccaga aagcgaccac ccaactttgg
     6721 ctgataatag cgtataaaca atgcatactt tgtacgttca aaatacaatg cagtagatat
     6781 atttatgcat attacatata atacatatca cataggaagc aacaggcgcg ttggactttt
     6841 aattttcgag gaccgcgaat ccttacatca cacccaatcc cccacaagtg atcccccaca
     6901 caccatagct tcaaaatgtt tctactcctt ttttactctt ccagattttc tcggactccg
     6961 cgcatcgccg taccacttca aaacacccaa gcacagcata ctaaatttcc cctctttctt
     7021 cctctagggt gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa gagaccgcct
     7081 cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc tttttcttga
     7141 aaattttttt ttttgatttt tttctctttc gatgacctcc cattgatatt taagttaata
     7201 aacggtcttc aatttctcaa gtttcagttt catttttctt gttctattac aacttttttt
     7261 acttcttgct cattagaaag aaagcatagc aatctaatct aagttttaat tacaaaatgg
     7321 gatcctctaa aggtgaagaa ttattcactg gtgttgtccc aattttggtt gaattagatg
     7381 gtgatgttaa tggtcacaaa ttttctgtct ccggtgaagg tgaaggtgat gctacttacg
     7441 gtaaattgac cttaaaattt atttgtacta ctggtaaatt gccagttcca tggccaacct
     7501 tagtcactac tttcggttat ggtgttcaat gttttgcgag atacccagat catatgaaac
     7561 aacatgactt tttcaagtct gccatgccag aaggttatgt tcaagaaaga actatttttt
     7621 tcaaagatga cggtaactac aagaccagag ctgaagtcaa gtttgaaggt gataccttag
     7681 ttaatagaat cgaattaaaa ggtattgatt ttaaagaaga tggtaacatt ttaggtcaca
     7741 aattggaata caactataac tctcacaatg tttacatcat ggctgacaaa caaaagaatg
     7801 gtatcaaagt taacttcaaa attagacaca acattgaaga tggttctgtt caattagctg
     7861 accattatca acaaaatact ccaattggtg atggtccagt cttgttacca gacaaccatt
     7921 acttatccac tcaatctgcc ttatccaaag atccaaacga aaagagagac cacatggtct
     7981 tgttagaatt tgttactgct gctggtatta cccatggtat ggatgaattg tacaaaagat
     8041 cttaaactag taggactatt tgcaaaggga agggatgcta aggtagaggg tgaacgttac
     8101 agaaaagcag gctgggaagc atatttgaga agatgcggcc agcaaaacta aaaaactgta
     8161 ttataagtaa atgcatgtat actaaactca c
//
