LOCUS       sequence-341159-        5906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression and purification of mature (no signal sequence) human 
            alpha1-antitrypsin wildtype variant M1V (UniProt P01009) from E. 
            coli BL21(DE3) cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5906)
  AUTHORS   Krishnan B, Hedstrom L, Hebert DN, Gierasch LM, Gershenson A
  TITLE     Expression and Purification of Active Recombinant Human Alpha-1 
            Antitrypsin (AAT) from Escherichia coli.
  JOURNAL   Methods Mol Biol. 2017;1639:195-209. doi: 
            10.1007/978-1-4939-7163-3_19.
  PUBMED    28752459
REFERENCE   2  (bases 1 to 5906)
  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: "Methods Mol
            Biol."; date: "2017"; volume: "1639:195-209. doi"; pages: " 
            10.1007/978-1-4939-7163-3_19"
COMMENT     Sequence Label: pEAT8-137 M1V
FEATURES             Location/Qualifiers
     source          1..5906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..111
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     345..362
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    377..516
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(442..461)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     602..624
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(618..809)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2267..2286
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2370..2389
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2370..2388
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2420..2442
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3721..3753
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     primer_bind     complement(3805..3823)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3819..3866
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4232..4237,4238..4254,4255..4260))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4287..4305)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4373..4477
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4478..4546,4547..5338)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4696..4715)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5509..5906,1..191)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
       61 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
      121 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      181 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      241 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
      301 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
      361 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
      421 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
      481 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
      541 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
      601 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
      661 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
      721 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
      781 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
      841 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
      901 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
      961 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     1021 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     1081 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     1141 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     1201 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     1261 atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg
     1321 gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc
     1381 cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg
     1441 cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga
     1501 caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg
     1561 cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt taggctggta agagccgcga
     1621 gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca
     1681 acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc
     1741 ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa
     1801 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     1861 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     1921 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     1981 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     2041 ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc cttatgcgac tcctgcatta
     2101 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     2161 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     2221 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     2281 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     2341 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg
     2401 gtttccctct agaaataatt ttgtttaact ttaagaagga gatataccat ggatccccag
     2461 ggagatgctg cccagaagac agatacatcc caccatgatc aggatcaccc aaccttcaac
     2521 aagatcaccc ccaacctggc tgagttcgcc ttcagcctat accgccagct ggcacaccag
     2581 tccaacagca ccaatatctt cttctcccca gtgagcatcg ctacagcctt tgcaatgctc
     2641 tccctgggga ccaaggctga cactcacgat gaaatcctgg agggcctgaa tttcaacctc
     2701 acggagattc cggaggctca gatccatgaa ggcttccagg aactcctccg taccctcaac
     2761 cagccagaca gccagctcca gctgaccacc ggcaatggcc tgttcctcag cgagggcctg
     2821 aagctagtgg ataagttttt ggaggacgtc aaaaagctgt accactcaga agccttcact
     2881 gtcaacttcg gggacaccga agaggccaag aaacagatca acgattacgt ggagaagggt
     2941 actcaaggga aaattgtgga tttggtcaag gagcttgaca gagacacagt ttttgctctg
     3001 gtgaattaca tcttctttaa aggcaaatgg gagagaccct ttgaagtcaa ggacaccgag
     3061 gaagaggact tccacgtgga ccaggtgacc accgtgaagg tgcctatgat gaagcgttta
     3121 ggcatgttta acatccagca ctgtaagaag ctgtccagct gggtgctgct gatgaaatac
     3181 ctgggcaatg ccaccgccat cttcttcctg cctgatgagg ggaaactaca gcacctggaa
     3241 aatgaactca cccacgatat catcaccaag ttcctggaaa atgaagacag aaggtctgcc
     3301 agcttacatt tacccaaact gtccattact ggaacctatg atctgaagag cgtcctgggt
     3361 caactgggca tcactaaggt cttcagcaat ggggctgacc tctccggggt cacagaggag
     3421 gcacccctga agctctccaa ggccgtgcat aaggctgtgc tgaccatcga cgagaaaggg
     3481 actgaagctg ctggggccat gtttttagag gccataccca tgtctatccc ccccgaggtc
     3541 aagttcaaca aaccctttgt cttcttaatg attgaacaaa ataccaagtc tcccctcttc
     3601 atgggaaaag tggtgaatcc cacccaaaaa taactgcctc tcgctcctca acccctcccc
     3661 tccatccctg gccccctccc tggatgacat taaagaaggg tcgcgaattc ccggggatcc
     3721 atggctagca tgactggtgg acagcaaatg ggtcggatcc ggctgctaac aaagcccgaa
     3781 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     3841 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga tatccacagg
     3901 acgggtgtgg tcgccatgat cgcgtagtcg atagtggctc caagtagcga agcgagcagg
     3961 actgggcggc ggccaaagcg gtcggacagt gctccgagaa cgggtgcgca tagaaattgc
     4021 atcaacgcat atagcgctag cagcacgcca tagtgactgg cgatgctgtc ggaatggacg
     4081 atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg
     4141 gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg
     4201 cgttagcaat ttaactgtga taaactaccg cattaaagct tatcgatgat aagctgtcaa
     4261 acatgagaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4321 catgataata atggtttctt agacgtcagg tgacactttt cggggaaatg tgcgcggaac
     4381 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4441 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4501 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4561 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4621 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4681 cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca
     4741 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4801 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4861 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4921 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4981 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
     5041 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     5101 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5161 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5221 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5281 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5341 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5401 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5461 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5521 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5581 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5641 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5701 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5761 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5821 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5881 gagataccta cagcgtgagc tatgag
//
