LOCUS       sequence-326335-        5807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  P. pastoris integration vector for the gene replacement of SEC13 
            fused to mEGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5807)
  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 5807)
  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-mEGFP
FEATURES             Location/Qualifiers
     source          1..5807
                     /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             join(583..585,586..588,589..1302)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(628..649)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(889..908)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1236..1257
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1940..1956)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1940..1956)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1953..1975)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1964..1980
                     /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(1988..1994,1995..2012,2013..2018))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2033..2054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2171..2188)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2342..2930)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2422..2441)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3101..3892,3893..3961))
                     /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     3724..3743
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             4289..5680
                     /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 ccatggtgag caagggcgag
      601 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      661 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
      721 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
      781 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
      841 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
      901 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
      961 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     1021 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     1081 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     1141 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     1201 aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1261 gccgccggga tcactctcgg catggacgag ctgtacaagt aaagcggccg taaaaggata
     1321 caaatataaa tacgttttaa ctcatttaac caattactat gtattctgtt attattgcta
     1381 ttaacgctca ttaacgtcta ctaacgttct tttaacagct tgttcctctt ttctttcctc
     1441 ttaagtctct cgacctttct acgatgcatt acttgagcca ttctctcata tctttccttt
     1501 tcttccttca aactttggcg gcgtttggtt tcttcaattt tgctttttct tatgctttct
     1561 ttctcctcct tgagttctct tatctttgcc tttatagctt gctcttcaag tcttttctgt
     1621 tttttctgtt cccaagtcag attgcctcct aaagatctca ctctcatggc atctttctgt
     1681 agcttccaat cttttccatt aacacgggtc cctcttcctt tatccttagg ggctataggg
     1741 ttgttatata tatccttctt tgaagtactg ctactgtcac tcatgatgta gtagtggttg
     1801 tgtagaatat caaaaggaaa aaaaaaatta tttcgtgaaa attcaaataa ggtattgttc
     1861 tcattaaggg ataaaaaaat accctttatt gccattgaaa agtattacat cggtactcat
     1921 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     1981 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     2041 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2101 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2161 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2221 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2281 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2341 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2401 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2461 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2521 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2581 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2641 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2701 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2761 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     2821 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     2881 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     2941 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3001 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3061 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3121 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3181 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3241 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3301 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3361 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3421 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3481 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3541 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3601 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3661 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3721 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3781 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     3841 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     3901 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     3961 tactcttcct ttttcaataa cgacaatcaa ataacctgat ctgccaaggc tccatcatat
     4021 ctggcctaga acagtttttt tttttcgatt atttgttcgt tcttgtggtg gttactcatt
     4081 ggcagaatcc cgaaaatcat gattagtaga tgaatgactc actttttgga taagctggcg
     4141 caaattgaaa catgtgaaaa aaaaaaaaaa ggattataaa aggtcagcga agcacagaac
     4201 tctgagataa gactaccttt ctttagctag gggagaatat tcgcaattga agagctcaaa
     4261 agcagtatat aacaagacta aggcaaacat gtcagacggc actcaaaaac tatggggtgg
     4321 gagattcact ggtgaaaccg atcctttgat gcacctttac aatgcgtctc ttccgtatga
     4381 ttataagatg tataaggcag atttagaagg aactaaagta tacacagcgg gcttgcagaa
     4441 gttgggtctt ctaacggaga cagaattggc aaagatccat gaaggtttgg ctgaaatcaa
     4501 aaaagaatgg gacgctgaca aatttgtccg tcatccaaac gacgaggata tccatactgc
     4561 gaatgaaaga cgtcttggtg aactaattgg ccgcgatatt gctggtaaag tccacaccgg
     4621 tagatcccgt aatgatcaag ttgttaccga tttgagaata tactgtcgtg acattgtcaa
     4681 tgacaccctc tttccagctt taaagggctt ggttgaagtt ctaattaaga gggccgaagg
     4741 tgagatagat gtcttaatgc caggctacac acatttacaa agggcacaac ctattagatg
     4801 gtctcattgg ttgagctctt atgcaacata cttcaccgaa gattacaaga gactgggtca
     4861 aatactacac agattgaatc aatcaccact gggtgcaggc gctcttgctg gtcatcctta
     4921 cggcattgat agagaatttt tggctgaagg tttgggtttc aatagtgtaa ttggtaactc
     4981 cttggttgct gtttctgata gagatttcat cgtggagttg atgttttggg gaactttgtt
     5041 catgaaccat atttctcgtt ttgctgaaga tttgattata tattgtacag cagaatttgg
     5101 tttcatacag ttgagcgacg cctattcaac aggttcttct ttaatgcctc agaagaagaa
     5161 tgcagactcg ttagagttgt taagaggtaa atccggtaga gtatttggtg atctgacagg
     5221 attcttgatg agtttgaagg gtatcccatc tacttatgat aaagacatgc aagaagacaa
     5281 agagccacta ttcgattgct taacaactgt agagcactcc atgctgattg ccacaggtgt
     5341 tatttctacc ttaactgtaa ataaggaaaa gatggaagct gctctcacga tggatatgct
     5401 agctaccgac ttggcagatt acttggtcag aaagggtgtt ccattcagag agactcatca
     5461 catatctggt gagtgtgtcg ctactgctga aagacttggt ctaagcggta ttgataaatt
     5521 aaccttggag cagtatcaaa agatcgattc gagattcgga caagatcttt ttgaaacttt
     5581 taactttgaa caaagcgttg aaagacgaga tgctactggt ggaaccgcta aatctgctgt
     5641 attgaagcaa ttggataatt tgaaatccca attaaattag cagatggcga cgttatgcgc
     5701 aagaatttca tcaggtctgg tacttctagg acttcatcaa cccttcttta aatacttatt
     5761 ttgttattag tattatattt tcgagattgt gctatgtata tatatac
//
