LOCUS       sequence-306491-        7275 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of 2AFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7275)
  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 7275)
  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: pMAL-C2X dAmp/Kana+-2AFP
FEATURES             Location/Qualifiers
     source          1..7275
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..80
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             81..1163
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(100..119)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1406..1411,1412..1427,1428..1434)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1442..1458
                     /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)."
     primer_bind     1447..1469
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1528..2628
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2602..2625
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2677..2688
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3112..3129
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3142..3159)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3142..3158)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3151..3173)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3337..3354)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3507..3593
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3685..3712
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3852..3871)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3863..4678
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             3863..4678
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3936..3955)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4949..5462)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5144..5165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5356..5375
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5573..6161
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6062..6081
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6315..6332
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6347..6489
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6415..6434)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6575..6597
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6591..6782)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
       61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcagta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1501 gcacttcacc aacaaggacc atagcatatg aaaatcgaag aaggtaaact ggtaatctgg
     1561 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
     1621 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
     1681 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
     1741 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
     1801 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
     1861 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
     1921 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
     1981 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
     2041 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
     2101 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
     2161 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     2221 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     2281 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     2341 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     2401 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     2461 ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg
     2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg
     2641 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaaggat ttcagaattc
     2701 ggatccatgc agtgtactgg cggtgcagac tgtacctcct gcaccggcgc ttgtaccggt
     2761 tgcggcaact gtccgaacgc tgtggcatgc actaactctc agcactgcgt taaagcgaac
     2821 gcgtgcaccg gttccactga ttgcaacacc gcgcaggctt gcactaactc caaagactgc
     2881 tttgaggcga acgcttgtac ggattccacc aattgttata aagcgacagc gtgcaccggg
     2941 tccggtaact gcccgaacgc agtggcttgc accggttcta ccgattgtaa caccgcacag
     3001 gcgtgcacta actctaagga ttgctttgaa gcgaacgcat gcacggattc caccaattgc
     3061 tacaaagcga ctgcttgtac caactcgcag cactgcgtta aggcgaacgc gcatcaccac
     3121 catcatcact aaaagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     3181 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     3241 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggca
     3301 gcttggctgt tttggcggat gagataagat tttcagcctg atacagatta aatcagaacg
     3361 cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga
     3421 ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca
     3481 tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     3541 cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg
     3601 gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat
     3661 aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc
     3721 tacaaactct tttgtttatt tttctaaata caggtacctt ctacggggtc tgacgctcag
     3781 tggaacgaaa actcacgtta agggattttg gtcatgaaca ataaaactgt ctgcttacat
     3841 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctctaggcc
     3901 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     3961 cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt
     4021 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     4081 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     4141 tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata
     4201 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     4261 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     4321 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     4381 gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt
     4441 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     4501 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     4561 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     4621 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga
     4681 attaattcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4741 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
     4801 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     4861 cccttataaa tcaaaagaat agaccgagat agggttgagt gccatggctg tcagaccaag
     4921 tttactcata tatactttag attgatttac cccggttgat aatcagaaaa gccccaaaaa
     4981 caggaagatt gtataagcaa atatttaaat tgtaaacgtt aatattttgt taaaattcgc
     5041 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     5101 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     5161 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     5221 tggcccacta cgtgaaccat cacccaaatc aagttttttg gggtcgaggt gccgtaaagc
     5281 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     5341 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     5401 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     5461 gtaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5521 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5581 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5641 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5701 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5761 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5821 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5881 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5941 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6001 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6061 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6121 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6181 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     6241 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     6301 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     6361 ttctccttac gcatctgtgc ggtatttcac accgcatata tggtgcactc tcagtacaat
     6421 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     6481 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     6541 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     6601 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc
     6661 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     6721 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     6781 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     6841 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     6901 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     6961 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     7021 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     7081 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     7141 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     7201 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca
     7261 acgctgcccg aaatt
//
