LOCUS       sequence-490438-        6303 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express engineered glycosylated eGFP in CHO cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6303)
  AUTHORS   Xing Y, Li Q, Otto EL, Campbell PG, Ren X
  TITLE     Glycosylation-enabled chemoselective growth factor engineering for 
            biomaterial functionalization.
  JOURNAL   Acta Biomater. 2025 Nov;207:221-232. doi: 
            10.1016/j.actbio.2025.09.057. Epub 2025 Oct 1.
  PUBMED    41043672
REFERENCE   2  (bases 1 to 6303)
  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: "Acta 
            Biomater."; date: "2025-11"; volume: "207:221-232. doi"; pages: " 
            10.1016/j.actbio.2025.09.057. Epub 2025 Oct 1"
COMMENT     Sequence Label: hVEGF165-eGFP-hVEGF165 glycosylation sequon-Hi...
FEATURES             Location/Qualifiers
     source          1..6303
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(985..987,988..1698)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             1759..1779
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1780..1797
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1894..1911)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1900..2124
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2170..2598
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2257..2276)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2467..2488
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2607..2627)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2612..2941
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2792..2927
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2854..2873
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3008..3802
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3062..3081)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3672..3691
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3976..4097
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4013..4032)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4067..4086
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4146..4162)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4146..4162)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4159..4181)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4170..4186
                     /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(4194..4200,4201..4218,4219..4224))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4239..4260
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4377..4394)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4548..5136)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4628..4647)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5307..6098,6099..6167))
                     /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     5930..5949
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6168..6272)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccaccatga attttctgct gagctgggtg cactggtccc tggccctgct gctgtacctg
      961 caccacgcca agtggtccca ggccgtgtct aagggcgagg agctgttcac aggcgtggtg
     1021 cccatcctgg tggagctgga cggcgatgtg aacggccaca agttttctgt gagcggagag
     1081 ggagagggcg acgcaaccta tggcaagctg acactgaagt tcatctgcac cacaggcaag
     1141 ctgcccgtgc cttggccaac actggtgacc acactgacct acggcgtgca gtgtttctcc
     1201 aggtatccag accacatgaa gcagcacgat ttctttaagt ctgccatgcc agagggatac
     1261 gtgcaggaga ggaccatctt ctttaaggac gatggcaatt ataagacaag agccgaggtg
     1321 aagttcgagg gcgacactct ggtgaacagg atcgagctga agggcatcga ctttaaggag
     1381 gatggcaata tcctgggcca caagctggag tacaactata attcccacaa cgtgtacatc
     1441 atggccgata agcagaagaa cggcatcaag gtgaacttca agatccgcca caatatcgag
     1501 gacggcagcg tgcagctggc cgatcactac cagcagaaca cacctatcgg cgacggaccc
     1561 gtgctgctgc ctgataatca ctatctgtcc acccagtctg ccctgagcaa ggacccaaac
     1621 gagaagcggg atcacatggt gctgctggag tttgtgaccg ccgccggcat cacactgggc
     1681 atggatgagc tgtacaaggg aggaggaggc agctccaaca tcaccatgtc taatatcaca
     1741 atgagcaaca tcaccatgga gaatctgtat ttccagggcc accaccacca ccaccactga
     1801 ggatccacta gtccagtgtg gtggaattct gcagatatcc agcacagtgg cggccgctcg
     1861 agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     1921 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     1981 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     2041 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     2101 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     2161 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2221 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2281 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2341 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2401 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2461 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2521 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2581 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     2641 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     2701 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2761 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     2821 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     2881 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     2941 aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc
     3001 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     3061 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     3121 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     3181 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     3241 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     3301 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     3361 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     3421 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     3481 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     3541 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     3601 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     3661 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     3721 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     3781 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     3841 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     3901 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     3961 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4021 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4081 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     4141 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4201 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4261 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4321 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4381 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4441 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4501 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4561 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4621 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4681 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4741 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4801 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4861 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4921 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4981 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5041 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5101 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5161 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5221 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5281 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5341 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5401 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5461 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5521 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5581 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5641 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5701 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5761 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5821 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5881 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5941 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6001 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6061 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6121 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6181 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6241 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6301 gtc
//
