LOCUS       sequence-329261-        5370 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of PilB Amber with a C-terminal histag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5370)
  AUTHORS   Katrina Forest
  TITLE     Forest Lab PilB Plasmids
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5370)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "unpublished"
COMMENT     Sequence Label: pUCP20 PilB113*L197D
FEATURES             Location/Qualifiers
     source          1..5370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     44..66
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1782..1799
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1799..2632)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pRO1600 from Pseudomonas aeruginosa "
                     /label=pRO1600 Rep
                     /translation="MASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     rep_origin      2646..2997
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     primer_bind     complement(3058..3077)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3177..3199
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3237..3255)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3323..3427
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3428..3496,3497..4288)
                     /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     complement(3646..3665)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4459..5047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4948..4967
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5201..5218
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5335..5356
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgaac gacagcatcc aactgagcgg tctgtcccga cagctcgtcc
      121 aagccaatct actcgacgaa aagaccgccc tgcaggccca gacgcaggcg cagcgcaaca
      181 agctgtcgct ggtgacgcac ctggtgcaga acaagctggt aagcggcctg gccctggccg
      241 aattgtccgc cgagcagttc ggcatcgcct attgcgacct gaacagcctg gacagggaaa
      301 gctttccgcg ggacgccatc agcgaaaagc tggtccgcca gcaccgggtc attcccctgt
      361 ggcggcgggg caacaagttg ttcgtaggca tctccgacgc cgccaaccac taggccatca
      421 acgacgtcca gttcagcact ggcctgacga ccgaggccat tctggtcgag gacgacaagc
      481 tcggccttgc catcgacaag ctattcgaga atgcaaccga cggcttggca ggtctcgatg
      541 acgtggacct ggaaggactg gatgtaggcg tcaaggaaac gtccggacaa gaagatactg
      601 gcgcggaggc agacgacgct cccgtcgtcc gcttcgtcaa caagatgctg gacgacgcta
      661 tcaaaggcgg gtcttccgac ttgcacttcg agccctacga gaagatctac cgggtgcgct
      721 tccgtaccga cggcatgctt cacgaagtgg ccaagccgcc gatccagttg gccagtcgta
      781 tctctgctcg tctcaaggta atggccggcc tggatatctc cgaacgacgc aagccgcagg
      841 acggacggat caagatgcgc gtgtcgaaga ccaagtccat cgacttccgc gtcaacaccc
      901 tgccgaccct gtggggcgag aagatcgtga tgcggatcct cgactcctcc agcgcgcaga
      961 tgggcatcga cgccctgggc tacgaggagg accagaagga actctacctg gccgcactca
     1021 agcagccgca gggcatgatc ctggtcaccg gccccaccgg ctcgggcaag acggtatcgc
     1081 tgtacaccgg cctgaacatc ctcaatacca ccgacatcaa catttccacc gccgaagacc
     1141 cggtggagat caacctggaa ggcatcaacc aggtcaacgt caatccgcgc cagggcatgg
     1201 acttctccca ggcgctgcgc gccttcctgc gccaggaccc ggacgtgatc atggtcggcg
     1261 agatccgcga cctggagacc gccgagatcg ccatcaaggc ggcgcagacc gggcatatgg
     1321 tgatgtccac cctgcacacc aacagcgccg ccgagaccct gacccgcctg ctgaacatgg
     1381 gcgtaccggc gttcaacctg gcgacctcgg tgaacctgat catcgcccag cgccttgcgc
     1441 gaaaactctg ttcgcactgc aagaaagagc acgacgtacc gaaggaaacc ctgcttcacg
     1501 aaggtttccc agaagagttg atcggcacct tcaagctgta ttcaccagtg ggctgcgacc
     1561 attgcaagaa cggttacaag ggccgtgtcg gtatttatga agtagttaaa aacaccccgg
     1621 ccctgcagcg gattatcatg gaggaaggca actccatcga gatcgccgag caagcccgca
     1681 aagaaggctt caacgatctg cgcacctcag gcctgctgaa agccatgcag gggatcacca
     1741 gcctggagga agtcaaccgc gtgaccaagg ataggccggg ccatcatcat caccatcatt
     1801 aagccagatc cagcggcatc tgggttagtc gagcgcgggc cgcttcccat gtctcaccag
     1861 ggcgagcctg tttcgcgatc tcagcatctg aaatcttccc ggccttgcgc ttcgctgggg
     1921 ccttacccac cgccttggcg ggcttcttcg gtccaaaact gaacaacaga tgtgtgacct
     1981 tgcgcccggt ctttcgctgc gcccactcca cctgtagcgg gctgtgctcg ttgatctgcg
     2041 tcacggctgg atcaagcact cgcaacttga agtccttgat cgagggatac cggccttcca
     2101 gttgaaacca ctttcgcagc tggtcaattt ctatttcgcg ctggccgatg ctgtcccatt
     2161 gcatgagcag ctcgtaaagc ctgatcgcgt gggtgctgtc catcttggcc acgtcagcca
     2221 aggcgtattt ggtgaactgt ttggtgagtt ccgtcaggta cggcagcatg tctttggtga
     2281 acctgagttc tacacggccc tcaccctccc ggtagatgat tgtttgcacc cagccggtaa
     2341 tcatcacact cggtcttttc cccttgccat tgggctcttg ggttaaccgg acttcccgcc
     2401 gtttcaggcg cagggccgct tctttgagct ggttgtagga agattcgata gggacacccg
     2461 ccatcgtcgc tatgtcctcc gccgtcactg aatacatcac ttcatcggtg acaggctcgc
     2521 tcctcttcac ctggctaata caggccagaa cgatccgctg ttcctgaaca ctgaggcgat
     2581 acgcggcctc gaccagggca ttgcttttgt aaaccattgg gggtgaggcc acgttcgaca
     2641 ttccttgtgt ataaggggac actgtatctg cgtcccacaa tacaacaaat ccgtcccttt
     2701 acaacaacaa atccgtccct tcttaacaac aaatccgtcc cttaatggca acaaatccgt
     2761 ccctttttaa actctacagg ccacggatta cgtggcctgt agacgtccta aaaggtttaa
     2821 aagggaaaag gaagaaaagg gtggaaacgc aaaaaacgca ccactacgtg gccccgttgg
     2881 ggccgcattt gtgcccctga aggggcgggg gaggcgtctg ggcaatcccc gttttaccag
     2941 tcccctatcg ccgcctgaga gggcgcagga agcgagtaat cagggtatcg aggcggattc
     3001 acccttggcg tccaaccagc ggcaccagcg gcgcctgaga ggatggtgca ctctcagtac
     3061 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     3121 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3181 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     3241 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     3301 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3361 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     3421 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     3481 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     3541 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     3601 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     3661 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     3721 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     3781 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     3841 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     3901 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     3961 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4021 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4081 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4141 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4201 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     4261 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     4321 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4381 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4441 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4501 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4561 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     4621 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4681 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4741 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4801 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4861 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4921 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4981 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5041 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5101 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5161 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     5221 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     5281 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     5341 gagttagctc actcattagg caccccaggc
//
