LOCUS       sequence-226680-        6708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  precursor outer membrane pore protein E, phoE with mutations Alanine
            249 to Cysteine and Glutamine 307 to Cysteine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6708)
  AUTHORS   Bariya P, Randall LL
  TITLE     Co-assembly of SecYEG and SecA fully restores the properties of the 
            native translocon.
  JOURNAL   J Bacteriol. 2018 Oct 1. pii: JB.00493-18. doi: 10.1128/JB.00493-18.
  PUBMED    30275279
REFERENCE   2  (bases 1 to 6708)
  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: "J 
            Bacteriol. 2018 Oct 1. pii: JB.00493-18. doi: 10.1128/JB.00493-18."
COMMENT     Sequence Label: pAL950
FEATURES             Location/Qualifiers
     source          1..6708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(1206..1224)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1220..1267
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1442..1447,1448..1464,1465..1470))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1497..1515)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1583..1687
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1688..1756,1757..2548)
                     /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(1906..1925)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2719..3307
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3208..3227
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3461..3478
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3493..3635
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3561..3580)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3721..3743
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3737..3928)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5240..6322)
                     /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     6284..6303
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6323..6400)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6606..6625
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gaaaaagagc actctggcat tagtggtgat
      121 gggcattgtg gcatctgcat ctgtacaggc tgcagaaata tataataaag acggtaataa
      181 actggatgtc tatggcaaag ttaaagccat gcattatatg agtgataacg ccagtaaaga
      241 tggcgaccag agttatatcc gttttggttt caaaggcgaa acacaaatta acgatcaact
      301 gactggttat ggtcgttggg aagcagagtt tgccggtaat aaagcagaga gtgatactgc
      361 acagcaaaaa acgcgtctcg cttttgccgg gttgaaatat aaagatttgg gttctttcga
      421 ttatggtcgt aacctggggg cgttgtatga cgtggaagcc tggaccgata tgttcccgga
      481 atttggtggc gattcctcgg cgcagaccga caactttatg accaaacgcg ccagcggtct
      541 ggcgacgtat cggaacaccg acttcttcgg cgttatcgat ggcctgaact taaccctgca
      601 atatcaaggg aaaaacgaaa accgcgacgt taaaaagcaa aacggcgatg gcttcggcac
      661 gtcattgacg tacgactttg gcggcagcga tttcgccatt agtggggcct ataccaactc
      721 agatcgcacc aacgagcaga acctgcaaag ccgtggcaca ggcaagcgtg cagaagcatg
      781 ggcaacaggt ctgaaatacg atgccaataa tatttatctg gcaactttct attctgaaac
      841 acgcaaaatg acgccaataa ctggcggctt tgccaataag acacagaact ttgaatgtgt
      901 cgctcaatac cagtttgact ttggtctgcg tccatcgctg ggttatgtct tatcgaaagg
      961 gaaagatatt gaaggtatcg gtgatgaaga tctggtcaat tatatcgacg tcggtgctac
     1021 gtattatttc aacaaaaata tgtcagcgtt tgttgattat aaaatcaact gtctggatag
     1081 cgataacaaa ttgaatatta ataatgatga tattgtcgcg gttggcatga cgtatcagtt
     1141 ttaatgaata ttgccggatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     1201 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     1261 ttttttgctg aaaggaggaa ctatatccgg atatcccgca agaggcccgg cagtaccggc
     1321 ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca
     1381 ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg
     1441 cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct
     1501 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     1561 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1621 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     1681 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     1741 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     1801 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     1861 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     1921 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     1981 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     2041 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     2101 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     2161 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     2221 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     2281 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     2341 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     2401 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     2461 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     2521 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     2581 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     2641 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2701 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2761 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2821 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     2881 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2941 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3001 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3061 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3121 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3181 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3241 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3301 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3361 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3421 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3481 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3541 catatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     3601 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     3661 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3721 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     3781 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     3841 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     3901 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     3961 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     4021 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     4081 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     4141 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     4201 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     4261 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4321 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4381 tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg
     4441 agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc
     4501 gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc
     4561 ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac
     4621 aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc
     4681 ataaatcgcc gtgacgatca gcggtccagt gatcgaagtt aggctggtaa gagccgcgag
     4741 cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa
     4801 cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc
     4861 tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat
     4921 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     4981 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     5041 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     5101 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     5161 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     5221 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5281 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     5341 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     5401 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     5461 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     5521 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5581 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5641 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5701 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     5761 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5821 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     5881 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     5941 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     6001 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     6061 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     6121 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     6181 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     6241 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     6301 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     6361 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     6421 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     6481 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6541 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6601 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     6661 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaat
//
