LOCUS       sequence-129418-        8073 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses N-His tagged nonglycosylated human serum transferrin 
            unable to release iron from the N-lobe or the C-lobe.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8073)
  AUTHORS   Halbrooks PJ, He QY, Briggs SK, Everse SJ, Smith VC, MacGillivray 
            RT, Mason AB
  TITLE     Investigation of the mechanism of iron release from the C-lobe of 
            human serum transferrin: mutational analysis of the role of a pH 
            sensitive triad.
  JOURNAL   Biochemistry. 2003 Apr 8;42(13):3701-7.
  PUBMED    12667060
REFERENCE   2  (bases 1 to 8073)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Biochemistry."; date: "2003-04-8"; volume: "42(13)"; pages: 
            "3701-7"
COMMENT     Sequence Label: pNUT N6His K206E/K534A hTFNG
FEATURES             Location/Qualifiers
     source          1..8073
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             190..207
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             208..219
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2509..2528)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2942..2958)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2942..2958)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2955..2977)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2966..2982
                     /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(2990..2996,2997..3014,3015..3020))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3035..3056
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3173..3190)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3344..3932)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3424..3443)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4103..4894,4895..4963))
                     /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     4726..4745
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4964..5068)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5136..5154
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5192..5214)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5314..5333
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5527..5549
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5541..5558
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5542..5558
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5779..5798
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(6525..7088)
                     /codon_start=1
                     /gene="Mus musculus Dhfr"
                     /product="mouse dihydrofolate reductase"
                     /label=DHFR
                     /translation="MVRPLNCIVAVSQNMGIGKNGDRPWPPLRNEFKYFQRMTTTSSVE
                     GKQNLVIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQP
                     ELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEY
                     PGVLSEVQEEKGIKYKFEVYEKKD"
     promoter        complement(7116..7473)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7130..7265
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        7282..7473
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     7430..7450
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 gtggtttgtg ctaaccacgt ctgtcttcac agctctgtgt tgccatgtgt gctgaacaaa
       61 aaataaaaat tattattgat tttatatttc gggtggcatc cctgtgaccc ctccccagtg
      121 atgaggctcg ccgtgggagc cctgctggtc tgcgccgtcc tagggctgtg tctggctgtc
      181 cctgataaac atcatcatca tcatcatatc gagggaaggg tccctgataa aactgtgaga
      241 tggtgtgcag tgtcggagca tgaggccact aagtgccaga gtttccgcga ccatatgaaa
      301 agcgtcattc catccgatgg tcccagtgtt gcttgtgtga agaaagcctc ctaccttgat
      361 tgcatcaggg ccattgcggc aaacgaagcg gatgctgtga cactggatgc aggtttggtg
      421 tatgatgctt acctggctcc caataacctg aagcctgtgg tggcagagtt ctatgggtca
      481 aaagaggatc cacagacttt ctattatgct gttgctgtgg tgaagaagga tagtggcttc
      541 cagatgaacc agcttcgagg caagaagtcc tgccacacgg gtctaggcag gtccgctggg
      601 tggaacatcc ccataggctt actttactgt gacttacctg agccacgtaa acctcttgag
      661 aaagcagtgg ccaatttctt ctcgggcagc tgtgcccctt gtgcggatgg gacggacttc
      721 ccccagctgt gtcaactgtg tccagggtgt ggctgctcca cccttaacca atacttcggc
      781 tactcgggag ccttcaagtg tctgaaggat ggtgctgggg atgtggcctt tgtcgagcac
      841 tcgactatat ttgagaactt ggcaaacaag gctgacaggg accagtatga gctgctttgc
      901 ctggacaaca cccggaagcc ggtagatgaa tacaaggact gccacttggc ccaggtccct
      961 tctcataccg tcgtggcccg aagtatgggc ggcaaggagg acttgatctg ggagcttctc
     1021 aaccaggccc aggaacattt tggcaaagac aaatcaaaag aattccaact attcagctct
     1081 cctcatggga aggacctgct gtttaaggac tctgcccacg ggtttttaaa agtccccccc
     1141 aggatggatg ccaagatgta cctgggctat gagtatgtca ctgccatccg gaatctacgg
     1201 gaaggcacat gcccagaagc cccaacagat gaatgcaagc ctgtgaagtg gtgtgcgctg
     1261 agccaccacg agaggctcaa gtgtgatgag tggagtgtta acagtgtagg gaaaatagag
     1321 tgtgtatcag cagagaccac cgaagactgc atcgccaaga tcatgaatgg agaagctgat
     1381 gccatgagct tggatggagg gtttgtctac atagcgggca agtgtggtct ggtgcctgtc
     1441 ttggcagaaa actacgataa gagcgataat tgtgaggata caccagaggc agggtatttt
     1501 gctgtagcag tggtgaagaa atcagcttct gacctcacct gggacaatct gaaaggcaag
     1561 aagtcctgcc atacggcagt tggcagaacc gctggctgga acatccccat gggcctgctc
     1621 tacaataaga tcaaccactg cagatttgat gaatttttca gtgaaggttg tgcccctggg
     1681 tctaagaaag actccagtct ctgtaagctg tgtatgggct caggcctaaa cctgtgtgaa
     1741 cccaacaaca aagagggata ctacggctac acaggcgctt tcaggtgtct ggttgagaag
     1801 ggagatgtgg cttttgtggc acaccagact gtcccacaga acactggggg aaaaaaccct
     1861 gatccatggg ctaagaatct gaatgaaaaa gactatgagt tgctgtgcct tgatggtacc
     1921 aggaaacctg tggaggagta tgcgaactgc cacctggcca gagccccgaa tcacgctgtg
     1981 gtcacacgga aagataagga agcttgcgtc cacaagatat tacgtcaaca gcagcaccta
     2041 tttggaagcg acgtaactga ctgctcgggc aacttttgtt tgttccggtc ggaaaccaag
     2101 gaccttctgt tcagagatga cacagtatgt ttggccaaac ttcatgacag aaacacatat
     2161 gaaaaatact taggagaaga atatgtcaag gctgttggta acctgagaaa atgctccacc
     2221 tcatcactcc tggaagcctg cactttccgt agaccttaat agctcgaggt agggctgcca
     2281 ccaaggtgaa gatgggaacg cagatgatcc atgagtttgc cctggtttca ctggcccaac
     2341 ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa
     2401 gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg
     2461 tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac
     2521 caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag
     2581 cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca
     2641 gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca
     2701 actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt
     2761 gaaccactgc tcccttccct gtccttctga ttttaaaata actataccag caggaggacg
     2821 tccagacaca gcataggcta cctggccatg cccaaccggt gggacatttg agttgcttgc
     2881 ttggcactgt cctctcatgc gttgggtcca ctcagtagat gcctgttgaa ttcgtaatca
     2941 tgtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3001 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3061 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3121 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3181 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3241 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3301 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3361 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3421 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3481 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3541 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3601 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3661 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3721 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3781 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3841 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3901 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3961 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4021 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4081 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4141 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4201 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4261 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4321 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4381 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4441 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4501 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4561 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4621 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4681 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4741 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4801 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4861 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4921 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4981 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5041 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     5101 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     5161 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     5221 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     5281 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
     5341 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     5401 gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     5461 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     5521 ggttttccca gtcacgacgt tgtaaaacga cggccagtgc caagcttgca tgcctgcagg
     5581 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     5641 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     5701 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     5761 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     5821 gccggtgatg ccggccacga tgcgtccggc gtagaggatc tctgacggaa ggaaagaagt
     5881 cagaaggcaa aaacgagagt aactccacag tagctccaaa ttctttataa gggtcaatgt
     5941 ccatgcccca aagccaccca aggcacagct tggaggcttg aacagtggga catgtacaag
     6001 agatgattag gcagaggtga aaaagttgca tggtgctggt gcgcagacca atttatgcct
     6061 acagcctcct aatacaaaga cctttaacct aatctcctcc cccagctcct cccagtcctt
     6121 aaacacacag tctttgaagt aggcctcaag gtcggtcgtt gacattgctg ggagtccaag
     6181 agtcctctta tgtaagacct tgggcaggat ctgatgggcg ttcacggtgg tctccatgca
     6241 acgtgcagag gtgaagcgaa gtgcacacgg accggcagat gagaaggcac agacggggag
     6301 accgcgtaaa gagaggtgcg ccccgtggtc ggctggaacg gcagacggag aaggggacga
     6361 gagagtccca agcggccccg cgaggggtcg tccgcgggat tcagcgccga cgggacgtaa
     6421 acaaaggacg tcccgcgaag gatctaaagc cagcaaaagt cccatggtct tataaaaatg
     6481 catagcttta ggaggggagc agagaacttg aaagcatctt cctgttagtc tttcttctcg
     6541 tagacttcaa acttatactt gatgcctttt tcctcctgga cctcagagag gacgcctggg
     6601 tattctggga gaagtttata tttccccaaa tcaatttctg ggaaaaacgt gtcactttca
     6661 aattcctgca tgatccttgt cacaaagagt ctgaggtggc ctggttgatt catggcttcc
     6721 tggtaaacag aactgcctcc gactatccaa accatgtcta ctttacttgc caattccggt
     6781 tgttcaataa gtcttaaggc atcatccaaa cttttggcaa gaaaatgagc tcctcgtggt
     6841 ggttctttga gttctctact gagaactata ttaattctgt cctttaaagg tcgattcttc
     6901 tcaggaatgg agaaccaggt tttcctaccc ataatcacca gattctgttt accttccact
     6961 gaagaggttg tggtcattct ttggaagtac ttgaactcgt tcctgagcgg aggccagggt
     7021 cggtctccgt tcttgccaat ccccatattt tgggacacgg cgacgatgca gttcaatggt
     7081 cgaaccatga tggcaattct agaatcgata agctttttgc aaaagcctag gcctccaaaa
     7141 aagcctcctc actacttctg gaatagctca gaggccgagg cggcctcggc ctctgcataa
     7201 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     7261 gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc
     7321 atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga
     7381 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga
     7441 cacacattcc acagctggtt ctttccgcct cagaaggtac cttggttttt aaaaccagcc
     7501 tggagtagag cagatgggtt aaggtgagtg acccctcagc cctggacatt cttagatgag
     7561 ccccctcagg agtagagaat aatgttgaga tgagttctgt tggctaaaat aatcaaggct
     7621 agtctttata aaactgtctc ctcttctcct agcttcgatc cagagagaga cctgggcgga
     7681 gctggtcgct gctcaggaac tccaggaaag gagaagctga ggttaccacg ctgcgaatgg
     7741 gtttacggag atagctggct ttccggggtg agttctcgta aactccagag cagcgatagg
     7801 ccgtaatatc ggggaaagca ctatagggac atgatgttcc acacgtcaca tgggtcgtcc
     7861 tatccgagcc agtcgtgcca aaggggcggt cccgctgtgc acactggcgc tccagggagc
     7921 tctgcactcc gcccgaaaag tgcgctcggc tctgccaagg acgcggggcg cgtgactatg
     7981 cgtgggctgg agcaaccgcc tgctgggtgc aaaccctttg cgcccggact cgtccaacga
     8041 ctataaagag ggcaggctgt cctctaagcg tca
//
