LOCUS       sequence-199442-        7584 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express a DNA helicase, a chimeric construct of Bacilllus 
            stearothermophilus PcrA and 2B domain of Staphylococcus aureus PcrA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7584)
  TITLE     Webb Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7584)
  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            "Unpublished"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: pET22B_PcrA_2Bchimera
FEATURES             Location/Qualifiers
     source          1..7584
                     /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)"
     CDS             2316..2333
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2386..2404)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2400..2447
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2484..2939
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2571..2590)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2781..2802
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2965..3069
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3070..3138,3139..3930)
                     /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     complement(3288..3307)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4101..4689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4590..4609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4843..4860
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4875..5014
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4940..4959)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5100..5122
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5116..5307)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6116..7198)
                     /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     7160..7179
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7199..7276)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7482..7501
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gaactttctg agcgaacagc tgctggcaca
      121 tctgaataaa gaacagcaag aagcagttcg taccaccgaa ggtccgctgc tgattatggc
      181 aggcgcaggt agcggtaaaa cccgtgttct gacccatcgt attgcatatc tgatggcaga
      241 aaaacatgtt gcaccgtgga atattctggc aattaccttt accaataaag ccgcacgtga
      301 aatgcgtgaa cgtgttcaga gcctgctggg tggtgcagca gaagatgttt ggattagcac
      361 ctttcatagc atgtgtgttc gtattctgcg tcgtgatatt gatcgcattg gtattaatcg
      421 caactttagc attctggacc cgaccgatca gctgagcgtt atgaaaacca ttctgaaaga
      481 aaaaaacatc gatccgaaaa aattcgagcc tcgtaccatt ctgggcacca ttagcgcagc
      541 aaaaaatgaa ctgctgcctc cggaacagtt tgcaaaacgt gcaagcacct attatgaaaa
      601 agttgtgtcc gatgtgtacc aagaatatca gcaacgtctg ctgcgtaatc atagcctgga
      661 ttttgatgat ctgattatga ccaccatcca gctgtttgat cgtgttccgg atgttctgca
      721 ttattatcag tacaaattcc agtacatcca catcgatgaa tatcaggata ccaatcgtgc
      781 acagtatacc ctggttaaaa aactggcaga acgctttcag aacatttgtg cagttggtga
      841 tgcagatcag agcatttatc gttggcgtgg tgcagatatt caaaacattc tgagctttga
      901 acgcgattat ccgaatgcca aagttattct gctggaacag aattatcgta gcaccaaacg
      961 cattctgcag gcagcaaatg aagtgattga acataatgtg aaccgtaaac cgaaacgtct
     1021 gtggaccgaa aatccggaag gtaaaccgat tctgtattat gaagcaatga acgaagcaga
     1081 tgaagcacag tttgttgcag gtcgtattcg tgaagcagtt gaacgtggtg aacgtcgtta
     1141 tcgtgatttt gcagttctgt atcgtaccaa tgcacagagc cgtgttatgg aagaaatgct
     1201 gctgaaagca aatatcccgt atcagattgt tggtggccag aaattctatg atcgcaaaga
     1261 aattaaagat ctgctgagct atctgcgcat tattgccaat agcaatgatg atattagcct
     1321 gcagcgtatt atcaatgttc cgaaacgtgg tgttggtccg agcagcgttg aaaaagtgca
     1381 gaattatgcc ctgcagaaca acatcagcat gtttgatgca ctgggtgaag cagatttcat
     1441 tggtctgagc aaaaaagtta cccaagaatg cctgaacttc tatgaactga ttcagtccct
     1501 gatcaaagaa caagaattcc tggaaatcca tgagatcgtt gatgaagtgc tgcagaaaag
     1561 cggttatcgc gaaatgctgg aacgtgaaaa taccctggaa agccgtagcc gtctggaaaa
     1621 tattgatgaa tttatgagcg tgccgaaaga ttatgaagaa aatacaccgc tggaagaaca
     1681 gagcctgatc aattttctga ccgatctgag cctggttgca gatatcgatg aagcaaatgg
     1741 caccgaacag gcagccgaag gtgatgccgt tatgctgatg accctgcatg ccgcaaaagg
     1801 tctggaattt ccggttgtgt ttctgattgg catggaagaa ggtatttttc cgcataatcg
     1861 ttccctggaa gatgatgatg aaatggaaga ggaacgtcgt ctggcctatg ttggtattac
     1921 ccgtgcagaa gaagaactgg ttctgaccag cgcacagatg cgtaccctgt ttggtaacat
     1981 tcagatgaat ccgcctagcc gttttctgaa tgaaattcct gcacatctgc tggaaaccgc
     2041 aagccgtcgt caggctggtg ccagccgtcc ggcagttagt cgtccgcagg caagcggtgc
     2101 cgttggtagc tggaaagtgg gtgatcgtgc aaatcatcgt aaatggggta ttggcaccgt
     2161 tgttagcgtt cgtggtggcg gtgatgatca agaactggat attgcatttc cgagcccgat
     2221 tggcattaaa cgcctgctgg caaaatttgc accgatcgaa aaagtgtaag gatccgaatt
     2281 cgagctccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc
     2341 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     2401 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     2461 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2521 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2581 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2641 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2701 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2761 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2821 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2881 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     2941 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3001 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3061 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3121 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3181 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3241 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3301 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3361 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3421 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3481 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3541 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3601 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3661 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3721 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3781 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3841 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3901 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3961 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     4021 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4081 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4141 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4201 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4261 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4321 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4381 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4441 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4501 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4561 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4621 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4681 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4741 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4801 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4861 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4921 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4981 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     5041 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5101 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     5161 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     5221 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5281 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5341 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5401 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5461 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5521 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5581 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5641 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5701 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5761 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     5821 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     5881 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     5941 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     6001 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     6061 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     6121 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6181 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     6241 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     6301 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     6361 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     6421 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     6481 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     6541 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     6601 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     6661 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     6721 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     6781 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     6841 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     6901 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     6961 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     7021 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     7081 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     7141 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     7201 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     7261 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     7321 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     7381 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     7441 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     7501 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     7561 atcgagatct cgatcccgcg aaat
//
