LOCUS       sequence-183401-        6134 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli Mechanosensitive Channel of Large Conductance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6134)
  TITLE     EcMscL
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6134)
  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: 
            "Unpublished"
COMMENT     Sequence Label: EcMscL
FEATURES             Location/Qualifiers
     source          1..6134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      20..608
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     509..528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     762..779
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    794..933
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(859..878)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1019..1041
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1035..1226)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2538..3620)
                     /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     3582..3601
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3621..3698)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3904..3923
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4007..4026
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4007..4025
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4026..4050
                     /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             4065..4087
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4106..4123
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4133..4150
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(4641..4659)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4655..4702
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4877..4882,4883..4899,4900..4905))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4932..4950)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5018..5122
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5123..5191,5192..5983)
                     /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(5341..5360)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
       61 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
      121 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
      181 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
      241 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
      301 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
      361 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
      421 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
      481 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
      541 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
      601 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
      661 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
      721 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
      781 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
      841 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
      901 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
      961 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     1021 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     1081 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     1141 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     1201 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     1261 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     1321 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     1381 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     1441 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     1501 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     1561 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     1621 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     1681 atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag
     1741 atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc
     1801 aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt
     1861 ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa
     1921 ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat
     1981 aaatcgccgt gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg
     2041 atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg
     2101 cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc
     2161 gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg
     2221 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     2281 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     2341 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     2401 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     2461 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     2521 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     2581 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     2641 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     2701 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     2761 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     2821 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     2881 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     2941 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     3001 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     3061 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     3121 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     3181 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     3241 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     3301 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     3361 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     3421 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     3481 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     3541 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     3601 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     3661 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     3721 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     3781 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     3841 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     3901 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     3961 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
     4021 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
     4081 aaggagatat accatgggca gcagccatca tcatcatcat cacagcagcg gcctggtgcc
     4141 gcgcggcagc catatgagca ttattaaaga atttcgcgaa tttgcgatgc gcgggaacgt
     4201 ggtggatttg gcggtgggtg tcattatcgg tgcggcattc gggaagattg tctcttcact
     4261 ggttgccgat atcatcatgc ctcctctggg cttattaatt ggcgggatcg attttaaaca
     4321 gtttgctgtc acgctacgcg atgcgcaggg ggatatccct gctgttgtga tgcattacgg
     4381 tgtcttcatt caaaacgtct ttgattttct gattgtggcc tttgccatct ttatggcgat
     4441 taagctaatc aacaaactga atcggaaaaa agaagaacca gcagccgcac ctgcaccaac
     4501 taaagaagaa gtattactga cagaaattcg tgatttgctg aaagagcaga ataaccgctc
     4561 ttaacaagcg cctgaaagca gaagaccagt ggatccggct gctaacaaag cccgaaagga
     4621 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     4681 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg
     4741 cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg
     4801 atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac
     4861 tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg agaattcttg
     4921 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     4981 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5041 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5101 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5161 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5221 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5281 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5341 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     5401 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5461 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5521 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     5581 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     5641 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     5701 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     5761 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5821 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     5881 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5941 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6001 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6061 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6121 gtcagacccc gtag
//
