LOCUS       sequence-307593-        8070 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 8070)
  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 8070)
  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 -2AFP-FliG
FEATURES             Location/Qualifiers
     source          1..8070
                     /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             4123..4140
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4161..4178)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4161..4177)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4170..4192)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4356..4373)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4526..4612
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4704..4731
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4750..4841
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4842..4910,4911..5702)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5060..5079)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5744..6257)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5939..5960)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6151..6170
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      6368..6956
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6857..6876
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7110..7127
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7142..7284
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7210..7229)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7370..7392
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7386..7577)
                     /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 ggatcctcta gaatgcagtg tactggcggt gcagactgta cctcctgcac cggcgcttgt
     2761 accggttgcg gcaactgtcc gaacgctgtg gcatgcacta actctcagca ctgcgttaaa
     2821 gcgaacgcgt gcaccggttc cactgattgc aacaccgcgc aggcttgcac taactccaaa
     2881 gactgctttg aggcgaacgc ttgtacggat tccaccaatt gttataaagc gacagcgtgc
     2941 accgggtccg gtaactgccc gaacgcagtg gcttgcaccg gttctaccga ttgtaacacc
     3001 gcacaggcgt gcactaactc taaggattgc tttgaagcga acgcatgcac ggattccacc
     3061 aattgctaca aagcgactgc ttgtaccaac tcgcagcact gcgttaaggc gaacggtggc
     3121 ggtggaagta tgagtaacct gacaggcacc gataaaagcg tcatcctgct gatgaccatt
     3181 ggcgaagacc gggcggcaga ggtgttcaag cacctctccc agcgtgaagt acaaaccctg
     3241 agcgctgcaa tggcgaacgt cacgcagatc tccaacaagc agctaaccga tgtgctggcg
     3301 gagtttgagc aagaagctga acagtttgcc gcactgaata tcaacgccaa cgattatctg
     3361 cgctcggtat tggtcaaagc tctgggtgaa gaacgtgccg ccagcctgct ggaagatatt
     3421 ctcgaaactc gcgataccgc cagcggtatt gaaacgctca actttatgga gccacagagc
     3481 gccgccgatc tgattcgcga tgagcatccg caaattatcg ccaccattct ggtgcatctg
     3541 aagcgcgccc aagccgccga tattctggcg ttgttcgatg aacgtctgcg ccacgacgtg
     3601 atgttgcgta tcgccacctt tggcggcgtg cagccagccg cgctggcgga gctgaccgaa
     3661 gtactgaatg gcttgctcga cggtcagaat ctcaagcgca gcaaaatggg cggcgtgaga
     3721 acggcagccg aaattatcaa cctgatgaaa actcagcagg aagaagccgt tattaccgcc
     3781 gtgcgtgaat tcgacggcga gctggcgcag aaaatcatcg acgagatgtt cctgttcgag
     3841 aatctggtgg atgtcgacga tcgcagcatt cagcgtctgt tgcaggaagt ggattccgaa
     3901 tcgctgttga tcgcgctgaa aggagccgag cagccactgc gcgagaaatt cttgcgcaat
     3961 atgtcgcagc gtgccgccga tattctgcgc gacgatctcg ccaaccgtgg tccggtgcgt
     4021 ctgtcgcagg tggaaaacga acagaaagcg attctgctga ttgtgcgccg ccttgccgaa
     4081 actggcgaga tggtaattgg cagcggcgag gatacctatg tccaccatca ccatcaccat
     4141 tgactgcagg caagcttggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     4201 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     4261 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcag
     4321 cttggctgtt ttggcggatg agataagatt ttcagcctga tacagattaa atcagaacgc
     4381 agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac
     4441 cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat
     4501 gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     4561 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg
     4621 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata
     4681 aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct
     4741 acaaactctt ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4801 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4861 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4921 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4981 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt ttcccaatga
     5041 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     5101 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5161 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5221 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5281 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5341 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5401 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5461 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5521 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5581 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5641 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5701 aactgtcaga ccaagtttac tcatatatac tttagattga tttaccccgg ttgataatca
     5761 gaaaagcccc aaaaacagga agattgtata agcaaatatt taaattgtaa acgttaatat
     5821 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     5881 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     5941 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     6001 cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt ttttggggtc
     6061 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     6121 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     6181 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     6241 gccgctacag ggcgcgtaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6301 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6361 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6421 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6481 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     6541 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6601 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6661 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6721 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6781 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6841 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6901 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6961 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     7021 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     7081 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     7141 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg
     7201 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     7261 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     7321 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     7381 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     7441 tcagcgtggt cgtgcagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     7501 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     7561 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     7621 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     7681 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     7741 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     7801 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     7861 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     7921 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     7981 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     8041 cgtggccagg acccaacgct gcccgaaatt
//
