LOCUS       sequence-104378-        5695 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of EGFP-TEV-S-tag hNSL1 for protein 
            localization and affinity purification (LAP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5695)
  TITLE     Cheeseman Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5695)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pIC79
FEATURES             Location/Qualifiers
     source          1..5695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1375..1395
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1408..1452
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    2483..2604
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2520..2539)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2574..2593
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2611..3066)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2748..2769)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2960..2979
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3093..3197
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3199..3556
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3199..3390
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3222..3242)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3407..3542
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3591..4385
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3591..4385
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3645..3664)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4255..4274
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4573..4592)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4617..4664
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4993..5581
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5482..5501
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccttcggc tacggcctgc agtgcttcgc ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggagggga tcggtggagc tccaccggtg gcggccgctc tagagagaat
     1381 ctttattttc agggcgccgc caaattcaaa gaaaccgctg ctgctaaatt cgaacgccag
     1441 cacatggaca gcggaggtgg aggtactagt ctcgagatgg cggggtttcc tgagttggtg
     1501 gtccttgacc ctccatggga caaggagctc gcggctggca cagagagcca ggccttggtc
     1561 tccgccactc cccgagaaga ctttcgggtg cgctgcacct cgaagcgggc tgtgaccgaa
     1621 atgctacaac tgtgcggccg cttcgtgcaa aagctcgggg acgctctgcc ggaggagatt
     1681 cgggagcccg ctctgcgaga tgcgcagtgg acttttgaat cagctgtgca agagaatatc
     1741 agcattaatg ggcaagcatg gcaggaagct tcagataatt gttttatgga ttctgacatc
     1801 aaagtacttg aagatcagtt tgatgaaatc atagtagata tagccacaaa acgtaagcag
     1861 tatcccagaa agatcctgga atgtgtcatc aaaaccataa aagcaaaaca agaaattctg
     1921 aagcagtacc accctgttgt acatccactg gacctaaaat atgaccctga tccagcccct
     1981 catatggaaa atttgaaatg cagaggggaa acagtagcaa aggagatcag tgaagccatg
     2041 aagtccttgc ctgcattaat tgaacaagga gagggatttt cccaagttct caggatgcag
     2101 cctgttatcc acctccagag gattcaccaa gaagtctttt ccagttgtca taggaaacca
     2161 gatgctaaac ctgagaactt tataacacag atagaaacca caccaacaga gactgcttcc
     2221 aggaaaacct ctgacgtggt actgaaaaga aagcaaacta aagactgccc ccagagaaaa
     2281 tggtatccat tgcggccaaa gaaaattaat cttgatacat gagaattctg cagtcgacgg
     2341 taccgcgggc ccgggatcca ccggatctag ataactgatc ataatcagcc ataccacatt
     2401 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     2461 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     2521 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     2581 gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat
     2641 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     2701 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     2761 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     2821 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     2881 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     2941 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     3001 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     3061 gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3121 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3181 attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg
     3241 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3301 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3361 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3421 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3481 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3541 cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac
     3601 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3661 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3721 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg
     3781 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     3841 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     3901 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     3961 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4021 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4081 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc
     4141 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     4201 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     4261 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     4321 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     4381 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     4441 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     4501 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag
     4561 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc
     4621 aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg
     4681 gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg
     4741 cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc
     4801 caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga
     4861 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4921 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4981 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5041 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5101 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     5161 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5221 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5281 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5341 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5401 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5461 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5521 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5581 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5641 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
