LOCUS       sequence-345150-        7335 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gfcD protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7335)
  AUTHORS   Larson MR, Biddle K, Gorman A, Boutom S, Rosenshine I, Saper MA
  TITLE     Escherichia coli O127 group 4 capsule proteins assemble at the outer
            membrane.
  JOURNAL   PLoS One. 2021 Nov 15;16(11):e0259900. doi: 
            10.1371/journal.pone.0259900. eCollection 2021.
  PUBMED    34780538
REFERENCE   2  (bases 1 to 7335)
  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: "PLoS One.";
            date: "2021-11-15"; volume: "16(11):e0259900. doi"; pages: " 
            10.1371/journal.pone.0259900. eCollection 2021"
COMMENT     Sequence Label: pMCSG26(gfcD)
FEATURES             Location/Qualifiers
     source          1..7335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1291..1482
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1476..1498)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1584..1723
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1639..1658
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1738..1755)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1909..2497)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1989..2008)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2668..3459,3460..3528))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3291..3310
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3529..3632)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3799..3818
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3942..3989)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3985..4003
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4056..4073)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4124..4141)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             6265..6285
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    6301..6325
                     /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     complement(6325..6344)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6326..6344)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6432..6451)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6657..6734
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(6735..7335,1..482)
                     /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(6754..6773)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
       61 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
      121 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
      181 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
      241 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
      301 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
      361 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
      421 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      481 gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg
      541 cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc
      601 gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc
      661 tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt
      721 gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa
      781 cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc
      841 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
      901 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
      961 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     1021 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     1081 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     1141 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     1201 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     1261 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     1321 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     1381 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     1441 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     1501 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     1561 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     1621 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg
     1681 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     1741 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1801 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1861 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1921 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1981 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2041 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2101 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2161 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2221 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2281 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2341 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2401 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2461 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2521 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     2581 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     2641 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     2701 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     2761 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     2821 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     2881 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     2941 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc
     3001 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     3061 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     3121 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     3181 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     3241 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     3301 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     3361 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     3421 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     3481 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     3541 tcaaattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3601 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     3661 ttgtaagcgt taatgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     3721 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     3781 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     3841 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     3901 cgcgtcccat tcgccaatcc ggatatagtt cctcctttca gcaaaaaacc cctcaagacc
     3961 cgtttagagg ccccaagggg ttatgctagt tattgctcag cggtggcagc agccaactca
     4021 gcttcctttc gggctttgtt agcagccgga tctcagtggt ggtggtggtg gtgctcgagt
     4081 gcggccgcaa gcttgtcgac ggagctcgaa ttcggatccg ttaatgatga tgatggtggt
     4141 gcccatggtg atggtgggca ctttgtccaa ccggtacttc tcgctctgcc gtcattggat
     4201 agagctggta tttgcgtgac agcatttgtc caccatcacg cgtcaatggt gtccacgaaa
     4261 ccaccgcgcg gttgcggtta ggtccaatgg tcatcaagtc gaacggaatc gagatataaa
     4321 aacctttact aaagccgcct tcgccgtagt catctttaga tacgttactg attgccgccc
     4381 atacccctac cgccacgccg ctgtcaaagc gtttggcgac gtcgatcgtt gcgcctttat
     4441 cttttgccag atattgccca acgctgagtt tcatcagtac gccgttgagc gtcggcgggt
     4501 tccagtaagc cgtcacgaaa cctgtgggtg tggaataatc ggtgaagcgc atcatgttgt
     4561 cccagtcacg ttgcttcacg tagttaacgt ccacacccag cgcccagctg gcatctagcg
     4621 ggcgataaag cagctcggaa ccgacaccgg cgtacatcgt ttccagataa ccgccataca
     4681 cctggccata gaaaccattg cctaagtcgg caaaatagtt cgcctgcaag ttgttgagat
     4741 aaacgtcatt gcgaacgtag tcacggatat gcgtgcgcac gcgggggagg gtagagtccg
     4801 cgggtaatag tgaagactta aacttgtcgt agttattgta aatattggtg aaaataccgc
     4861 catcaagcag caggtggtcg gtaaaccagt agcgggcgct ggacatcagc cccagctgga
     4921 acatatagaa atcttccggg ccgcccagcg actgtgaaag tgttgggttg aaagagtagc
     4981 taaagcgatc ttcacgaata cggtagcccc gaccaaaggc agaaagatct tctgcttcaa
     5041 cacgttgttg ttgcagttgc tctgatttac caggcgctgt accggccagc tgtttgcgca
     5101 ggctggctac gtcggtttcg gtagtcacca tcgccatatg ctcgcgcttt tgcgtcacgc
     5161 taatcttctc aacgccttgc ggcaggttat tcaccagaat ccggttggca cgatcgaccg
     5221 cttcacgaga gtcacggtat ttgtattgct caccagacat atacagcgtc ttatcgcgca
     5281 gctgaatttc tggcgcgtca aagcccgcgt tatacttcag ggcggtaagt tggtttgcta
     5341 ccgtggtgta ctgcaatcct tcagattcag gcgcaggttg atatgccggt tttggcgtat
     5401 cgcgcagggc agggcgcaga tcgttgaaat tggtccgtaa agtgaaacca aacatcaacg
     5461 tgttaccgcg ttcataactg aggttgaggt ctgcccagct ggcagcgcga taaactgcac
     5521 cgacgttgaa atggcttgcc tgaggcagtt tgccagcgaa atcattctgg taattgttgc
     5581 cgtcgtattc gagtttcaga cgcaggggat tccacggcgt ttgatactca atgccgccaa
     5641 agatggaagc cgggccacga aagatatcgc taaagctgat atcgcccgca tcgtgagact
     5701 ctgcacgatg acaatattta tcgctcaccc ggcaaaacgg gttggtaata ttgcccgcat
     5761 taccggcgta gccccatgcc atcccgaggg tgaaatcaaa tggccccgct tgcttgctgg
     5821 ccaccaaata ttcaccgtca aacagacccg taccagcaat atcacgttta ccaaacgcca
     5881 cttgcggtag caagtaacct tcttcccaca ggcgaagttt aaaatcgaaa gatttgtctt
     5941 tgtatgactg atcgccgctg aaatcttccc actggctata tttgcgtgtg cgcacatccg
     6001 tataacgaat ggtgccttcc agccatggga acagcgccac ggacgtggag tagaaacggt
     6061 attgatcgtt atcccggtag ttgacgctga attcaccttc cggtgcgatg cgcgcatttg
     6121 gcatctgcaa caatcctgtg ccgccaaagt ctgattgcga cgaccccagc ggatccgggt
     6181 aggttaatac ttcagcatga caggcactgg agacggcaat ggccaggcag cttaaaagat
     6241 aagagttctt cttcatggga gagactcctt cttaaagtta aacaaaatta tttctagagg
     6301 ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag
     6361 atcgatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
     6421 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
     6481 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
     6541 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
     6601 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca
     6661 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     6721 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     6781 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     6841 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     6901 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     6961 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     7021 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     7081 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     7141 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     7201 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     7261 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     7321 tgcgtctggc tggct
//
