LOCUS       sequence-326334-        5798 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  P. pastoris integration vector for the gene replacement of SEC13 
            fused to msGFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5798)
  AUTHORS   Valbuena FM, Fizgerald I, Strack RL, Andruska N, Smith L, Glick BS
  TITLE     A photostable monomeric superfolder GFP.
  JOURNAL   Traffic. 2020 May 15. doi: 10.1111/tra.12737.
  PUBMED    32415747
REFERENCE   2  (bases 1 to 5798)
  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: "Traffic. 
            2020 May 15. doi: 10.1111/tra.12737."
COMMENT     Sequence Label: pUC19-PpSEC13-msGFP2
FEATURES             Location/Qualifiers
     source          1..5798
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     180..202
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     194..211
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     195..211
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    212..268
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     regulatory      577..586
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             583..1290
                     /codon_start=1
                     /product="photostable monomeric superfolder GFP (Valbuena 
                     et al., 2020)"
                     /label=msGFP2
                     /translation="MDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITGGSGGS"
     primer_bind     complement(625..646)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1233..1254
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1931..1947)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1931..1947)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1944..1966)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1955..1971
                     /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(1979..1985,1986..2003,2004..2009))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2024..2045
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2162..2179)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2333..2921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2413..2432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3092..3883,3884..3952))
                     /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     3715..3734
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             4280..5671
                     /codon_start=1
                     /gene="S. cerevisiae ARG4"
                     /product="argininosuccinate lyase, required for arginine 
                     biosynthesis"
                     /label=ARG4
                     /note="yeast auxotrophic marker"
                     /translation="MSDGTQKLWGGRFTGETDPLMHLYNASLPYDYKMYKADLEGTKVY
                     TAGLQKLGLLTETELAKIHEGLAEIKKEWDADKFVRHPNDEDIHTANERRLGELIGRDI
                     AGKVHTGRSRNDQVVTDLRIYCRDIVNDTLFPALKGLVEVLIKRAEGEIDVLMPGYTHL
                     QRAQPIRWSHWLSSYATYFTEDYKRLGQILHRLNQSPLGAGALAGHPYGIDREFLAEGL
                     GFNSVIGNSLVAVSDRDFIVELMFWGTLFMNHISRFAEDLIIYCTAEFGFIQLSDAYST
                     GSSLMPQKKNADSLELLRGKSGRVFGDLTGFLMSLKGIPSTYDKDMQEDKEPLFDCLTT
                     VEHSMLIATGVISTLTVNKEKMEAALTMDMLATDLADYLVRKGVPFRETHHISGECVAT
                     AERLGLSGIDKLTLEQYQKIDSRFGQDLFETFNFEQSVERRDATGGTAKSAVLKQLDNL
                     KSQLN"
ORIGIN
        1 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc
       61 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
      121 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
      181 ccagtcacga cgttgtaaaa cgacggccag tgaattcgag ctcggtaccc ggggatcctc
      241 tagagtcgac ctgcaggcat gcaagctttt caaggacatt ccgactgggt cagagatgtg
      301 gcatggtctc cttctcgttt atcaaagtca tacattgcca ctgcctcaca agatcgtaca
      361 gtattgattt ggactaaaga tggaaaatct aacaaatggg aaaaacagcc tttaacaaag
      421 gaaaaattcc ccgatgtttg ttggagagcc agctggtctc ttagtggaaa tgtattggcc
      481 atttcgggtg gtgataataa ggtcactttg tggaaggaaa acatccaggg caaatgggag
      541 tccgctggcg aagtcgatca atacgatcca ccggtcgcca ccatggatag cactgagagc
      601 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
      661 ttcagcgtgc gcggcgaggg cgagggcgat gccaccaacg gcaagctgac cctgaagttc
      721 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
      781 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
      841 gccatgcccg aaggctacgt ccaggagcgc accatcacct tcaaggacga cggcacctac
      901 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
      961 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1021 agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag
     1081 atccgccaca acgtggagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1141 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccaag
     1201 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1261 gccgggatca ctggaggcag tggcggttct taaagcggcc gtaaaaggat acaaatataa
     1321 atacgtttta actcatttaa ccaattacta tgtattctgt tattattgct attaacgctc
     1381 attaacgtct actaacgttc ttttaacagc ttgttcctct tttctttcct cttaagtctc
     1441 tcgacctttc tacgatgcat tacttgagcc attctctcat atctttcctt ttcttccttc
     1501 aaactttggc ggcgtttggt ttcttcaatt ttgctttttc ttatgctttc tttctcctcc
     1561 ttgagttctc ttatctttgc ctttatagct tgctcttcaa gtcttttctg ttttttctgt
     1621 tcccaagtca gattgcctcc taaagatctc actctcatgg catctttctg tagcttccaa
     1681 tcttttccat taacacgggt ccctcttcct ttatccttag gggctatagg gttgttatat
     1741 atatccttct ttgaagtact gctactgtca ctcatgatgt agtagtggtt gtgtagaata
     1801 tcaaaaggaa aaaaaaaatt atttcgtgaa aattcaaata aggtattgtt ctcattaagg
     1861 gataaaaaaa taccctttat tgccattgaa aagtattaca tcggtactca tcaagcttgg
     1921 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     1981 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     2041 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     2101 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     2161 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     2221 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2281 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2341 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2401 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2461 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2521 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2581 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2641 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2701 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2761 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2821 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2881 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2941 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3001 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3061 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3121 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3181 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3241 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3301 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3361 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3421 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3481 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3541 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3601 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3661 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3721 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3781 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3841 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3901 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3961 tttttcaata acgacaatca aataacctga tctgccaagg ctccatcata tctggcctag
     4021 aacagttttt ttttttcgat tatttgttcg ttcttgtggt ggttactcat tggcagaatc
     4081 ccgaaaatca tgattagtag atgaatgact cactttttgg ataagctggc gcaaattgaa
     4141 acatgtgaaa aaaaaaaaaa aggattataa aaggtcagcg aagcacagaa ctctgagata
     4201 agactacctt tctttagcta ggggagaata ttcgcaattg aagagctcaa aagcagtata
     4261 taacaagact aaggcaaaca tgtcagacgg cactcaaaaa ctatggggtg ggagattcac
     4321 tggtgaaacc gatcctttga tgcaccttta caatgcgtct cttccgtatg attataagat
     4381 gtataaggca gatttagaag gaactaaagt atacacagcg ggcttgcaga agttgggtct
     4441 tctaacggag acagaattgg caaagatcca tgaaggtttg gctgaaatca aaaaagaatg
     4501 ggacgctgac aaatttgtcc gtcatccaaa cgacgaggat atccatactg cgaatgaaag
     4561 acgtcttggt gaactaattg gccgcgatat tgctggtaaa gtccacaccg gtagatcccg
     4621 taatgatcaa gttgttaccg atttgagaat atactgtcgt gacattgtca atgacaccct
     4681 ctttccagct ttaaagggct tggttgaagt tctaattaag agggccgaag gtgagataga
     4741 tgtcttaatg ccaggctaca cacatttaca aagggcacaa cctattagat ggtctcattg
     4801 gttgagctct tatgcaacat acttcaccga agattacaag agactgggtc aaatactaca
     4861 cagattgaat caatcaccac tgggtgcagg cgctcttgct ggtcatcctt acggcattga
     4921 tagagaattt ttggctgaag gtttgggttt caatagtgta attggtaact ccttggttgc
     4981 tgtttctgat agagatttca tcgtggagtt gatgttttgg ggaactttgt tcatgaacca
     5041 tatttctcgt tttgctgaag atttgattat atattgtaca gcagaatttg gtttcataca
     5101 gttgagcgac gcctattcaa caggttcttc tttaatgcct cagaagaaga atgcagactc
     5161 gttagagttg ttaagaggta aatccggtag agtatttggt gatctgacag gattcttgat
     5221 gagtttgaag ggtatcccat ctacttatga taaagacatg caagaagaca aagagccact
     5281 attcgattgc ttaacaactg tagagcactc catgctgatt gccacaggtg ttatttctac
     5341 cttaactgta aataaggaaa agatggaagc tgctctcacg atggatatgc tagctaccga
     5401 cttggcagat tacttggtca gaaagggtgt tccattcaga gagactcatc acatatctgg
     5461 tgagtgtgtc gctactgctg aaagacttgg tctaagcggt attgataaat taaccttgga
     5521 gcagtatcaa aagatcgatt cgagattcgg acaagatctt tttgaaactt ttaactttga
     5581 acaaagcgtt gaaagacgag atgctactgg tggaaccgct aaatctgctg tattgaagca
     5641 attggataat ttgaaatccc aattaaatta gcagatggcg acgttatgcg caagaatttc
     5701 atcaggtctg gtacttctag gacttcatca acccttcttt aaatacttat tttgttatta
     5761 gtattatatt ttcgagattg tgctatgtat atatatac
//
