LOCUS       sequence-306509-        6587 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of 2AFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6587)
  AUTHORS   Jiang Z, Jin X, Li Y, Liu S, Liu XM, Wang YY, Zhao P, Cai X, Liu Y, 
            Tang Y, Sun X, Liu Y, Hu Y, Li M, Cai G, Qi X, Chen S, Du LL, He W
  TITLE     Genetically encoded tags for direct synthesis of EM-visible gold 
            nanoparticles in cells.
  JOURNAL   Nat Methods. 2020 Sep;17(9):937-946. doi: 10.1038/s41592-020-0911-z.
            Epub 2020 Aug 10.
  PUBMED    32778831
REFERENCE   2  (bases 1 to 6587)
  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"; doi: 
            "10.1038/s41592-020-0911-z"; journalName: "Nat Methods"; date: 
            "2020-09"; volume: "17"; issue: "9"; pages: "937-946"
COMMENT     Sequence Label: pcDNA3.1-GFP-2AFP-KDEL
FEATURES             Location/Qualifiers
     source          1..6587
                     /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"
     regulatory      917..926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     923..982
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             join(986..988,989..1699)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNVEDGSVQLADHDQQNTPIGDGPVLLPDNHYLSTQSALSKDPNERRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1028..1049)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1289..1308)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1636..1657
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2178..2195)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2184..2408
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2454..2882
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2541..2560)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2751..2772
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2891..2911)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2896..3225
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3076..3211
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3138..3157
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3292..4086
                     /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(3346..3365)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3956..3975
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4260..4381
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4297..4316)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4351..4370
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4430..4446)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4430..4446)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4443..4465)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4454..4470
                     /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(4478..4484,4485..4502,4503..4508))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4523..4544
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4661..4678)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4832..5420)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4912..4931)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5591..6382,6383..6451))
                     /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     6214..6233
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6452..6556)
                     /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 gtttaaactt aagcttgcca ccatggagac agacacactc ctgttatggg tactgctgct
      961 ctgggttcca ggttccactg gtgacgtgag caagggcgag gagctgttca ccggggtggt
     1021 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1081 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1141 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     1201 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1261 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1321 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1381 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     1441 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacgtcga
     1501 ggacggcagc gtgcagctcg ccgaccacga ccagcagaac acccccatcg gcgacggccc
     1561 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     1621 cgagaggcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     1681 catggacgag ctgtacaagc tgcagagccg aatgcagtgt actggcggtg cagactgtac
     1741 ctcctgcacc ggcgcttgta ccggttgcgg caactgtccg aacgctgtgg catgcactaa
     1801 ctctcagcac tgcgttaaag cgaacgcgtg caccggttcc actgattgca acaccgcgca
     1861 ggcttgcact aactccaaag actgctttga ggcgaacgct tgtacggatt ccaccaattg
     1921 ttataaagcg acagcgtgca ccgggtccgg taactgcccg aacgcagtgg cttgcaccgg
     1981 ttctaccgat tgtaacaccg cacaggcgtg cactaactct aaggattgct ttgaagcgaa
     2041 cgcatgcacg gattccacca attgctacaa agcgactgct tgtaccaact cgcagcactg
     2101 cgttaaggcg aacggcagct ccagtaagga cgagctgtga ctcgagtcta gagggcccgt
     2161 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2221 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2281 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2341 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2401 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     2461 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2521 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2581 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2641 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2701 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2761 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2821 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2881 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     2941 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3001 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3061 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3121 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3181 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     3241 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     3301 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3361 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3421 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     3481 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3541 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3601 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3661 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3721 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3781 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     3841 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3901 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3961 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4021 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4081 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4141 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4201 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4261 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4321 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4381 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     4441 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4501 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4561 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4621 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4681 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4741 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4801 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4861 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4921 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4981 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5041 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5101 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5161 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5221 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5281 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5341 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5401 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5461 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5521 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5581 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5641 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5701 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5761 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5821 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5881 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5941 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6001 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6061 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6121 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6181 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6241 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6301 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6361 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6421 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6481 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6541 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
