LOCUS       sequence-322680-        5043 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a ST6GAL1 mutant with 20 amino acids in its TMD in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5043)
  AUTHORS   Sun X, Mahajan D, Chen B, Song Z, Lu L
  TITLE     A quantitative study of the Golgi retention of glycosyltransferases.
  JOURNAL   J Cell Sci. 2021 Oct 15;134(20). pii: 272560. doi: 
            10.1242/jcs.258564. Epub 2021 Oct 21.
  PUBMED    34533190
REFERENCE   2  (bases 1 to 5043)
  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: "J Cell 
            Sci."; date: "2021-10-15"; volume: "134(20). pii: 272560. doi"; 
            pages: " 10.1242/jcs.258564. Epub 2021 Oct 21"
COMMENT     Sequence Label: TMD20
FEATURES             Location/Qualifiers
     source          1..5043
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..355
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        356..559
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     510..530
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      973..982
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(979..981,982..984,985..1698)
                     /codon_start=1
                     /product="monomeric GFP variant optimized for use in 
                     oxidizing environments such as the secretory pathway 
                     (Costantini et al., 2015)"
                     /label=moxGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFISTTGKLPVPWPTLVTTLTYGVQSFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1024..1045)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    1820..1941
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1857..1876)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1911..1930
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1948..2403)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2085..2106)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2297..2316
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2430..2534
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2536..2893
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2536..2727
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2559..2579)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2744..2879
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2928..3722
                     /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             2928..3722
                     /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(2982..3001)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3592..3611
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3910..3929)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3954..4001
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4330..4918
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4819..4838
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
       61 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      121 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      181 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatatgc caagtacgcc
      241 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      301 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      361 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
      421 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      481 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
      541 ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggactca
      601 gatctcgagg ccaccatgat tcacaccaac ctgaagaaaa agttcagctg ctgcgtacta
      661 gtcgcagtac tgtttcttct gtttgcagtg atatgtgtgt ggaaggaaaa gaagaaaggg
      721 agttactatg attcctttaa attgcaaacc aaggaattcc aggtgttaaa gagtctgggg
      781 aaattggcca tggggtctga ttcccagtct gtatcctcaa gcagcaccca ggacccccac
      841 aggggccgcc agaccctcgg cagtctcaga ggcctagcca aggccaaacc agaggcctcc
      901 ttccaggtgt ggaacaagga cagctcttcc aaaaacctta tccctaggct gcaaaagatc
      961 tgggcaccgg tcgccaccat ggtgtccaag ggcgaggagc tgttcaccgg ggtggtgccc
     1021 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tctccgtgcg gggcgagggc
     1081 gagggcgatg ccaccaacgg caagctgacc ctgaagttca tcagcaccac cggcaagctg
     1141 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagag cttctcccgc
     1201 taccccgacc acatgaagcg ccacgacttc ttcaagagcg ccatgcccga aggctacgtc
     1261 caggagcgca ccatctcctt caaggacgac ggcacctaca agacccgcgc cgaggtgaag
     1321 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1381 ggcaacatcc tggggcacaa gctggagtac aacttcaact cccacaacgt ctatatcacc
     1441 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa cgtggaggac
     1501 ggctccgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1561 ctgctgcccg acaaccacta cctgtccacc cagtccaagc tgtccaaaga ccccaacgag
     1621 aagcgcgatc acatggtcct tctggaattc gtgaccgccg ccgggatcac tcacggcatg
     1681 gacgagctgt acaagtaagc ggccgcgact ctagatcata atcagccata ccacatttgt
     1741 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     1801 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     1861 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     1921 caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     1981 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     2041 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     2101 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     2161 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     2221 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     2281 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     2341 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     2401 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2461 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2521 gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     2581 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2641 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2701 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     2761 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     2821 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     2881 aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     2941 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3001 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3061 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
     3121 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3181 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3241 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3301 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3361 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     3421 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
     3481 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     3541 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     3601 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     3661 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     3721 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     3781 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     3841 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg
     3901 gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat
     3961 aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     4021 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     4081 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     4141 cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt
     4201 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4261 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4321 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4381 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4441 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     4501 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4561 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4621 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4681 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4741 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4801 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4861 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4921 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4981 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc attagttatt
     5041 aat
//
