LOCUS       sequence-456000-        6593 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression in BL21(DE3) of T4 Dda-like helicase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6593)
  TITLE     Escherichia phage T4 Expression Constructs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6593)
  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            "Unpublished"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: pET28b-T4Dda
FEATURES             Location/Qualifiers
     source          1..6593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1534..1556
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1571..1595
                     /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(1595..1614)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1596..1614)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1698..1717)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1923..2000
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2001..3083
                     /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(2020..2039)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3892..4083
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4077..4099)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4185..4324
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4240..4259
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4339..4356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4510..5098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4590..4609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5209..5228)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5220..6035
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5220..6035
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5293..5312)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6127..6582)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6264..6285)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6476..6495
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccctattat acataaaaca catcataacg accacgggtg
      241 acaccaacat aaagaagttg ttgagctaat tcaacatctg cataatgaat acaaggcgta
      301 taaatgaaag cacggtctac agacataccc tgcgctttat ggaatgttga tgcaggaagt
      361 gctttcactt tactaaactg tgatttagca tcccaaaaat cactccacgg agcttttccg
      421 cctttgttcc aatttttata agtttctgct gttttaccta aaaataggtt aaacttatac
      481 aattcttcgt cagatgaaat tattttaatc ttttcacgat aatattcatc atcgccataa
      541 gtttctactg ttaaatccca atgacgaatt agatattctc caggaacacc acgggcttta
      601 acaaacgttg atgtatactc tgcttctata atacgaacta attgtccgtt attaaaaata
      661 atttctgaca caggctttcc atcaatttta tatgttttaa ataatggttc ctgcattaca
      721 ataatttcac cgacaataaa atctttatca gtttcaaaaa tctttttacg aataatgcta
      781 tttaacttgt caacagattt attcgtaaat gccattacgc gattttcaaa caaatcatct
      841 agtgatttga cgattgaaaa ataatttacc ataaaatcgc gtaaagcggt atcaccagta
      901 aatccacgta ctccatgccc gtcaacaact ttatcataaa tccacttacc gttgcgaacg
      961 tcagtagcta catcaataat aggagcatta ctgcgtttaa cttcagtgag ttcacactga
     1021 taaaaatctt tatgtgtaaa gaatggactg atataagcag tattttctcc tgggtcaaca
     1081 ggtctgattt gcttattatc ccctattcca attatagtac accaaggtgg aatagttgaa
     1141 agcagaattt taaatagctt tctatcatac attgacactt cgtcgcagat taatactctg
     1201 catttggcta aatcaggtac ttctttttgt tcaaaaagaa cattttcttc ataggttact
     1261 gggttaattt taagaatact atgaatagta ctcgcttctt tccctgatag ttttgaaaga
     1321 atctttttag ctgcatgtgt aggagctgct aaaataatac cagtttcacc cgtagatatt
     1381 aaagcttcaa tgatgaactt agtaagagta gtcttaccgg taccagcagg tccattaata
     1441 gttacatgat gtttcttttc tttaatagcc ttcataacaa tgttaaaggc atttttctgg
     1501 ccttcggtca aatcatcaaa tgtcatatgt atatctcctt cttaaagtta aacaaaatta
     1561 tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg
     1621 cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac
     1681 aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca
     1741 cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg
     1801 actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct
     1861 caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc
     1921 cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
     1981 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
     2041 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
     2101 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
     2161 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
     2221 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
     2281 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
     2341 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
     2401 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
     2461 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
     2521 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
     2581 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
     2641 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
     2701 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
     2761 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
     2821 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
     2881 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     2941 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     3001 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     3061 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     3121 gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg
     3181 gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta
     3241 ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg
     3301 acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc
     3361 gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg
     3421 gccccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt
     3481 tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc
     3541 tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa
     3601 agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga
     3661 tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc
     3721 tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt
     3781 tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc
     3841 atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa
     3901 caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg
     3961 gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac
     4021 cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc
     4081 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     4141 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag
     4201 tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac
     4261 tgagagtgca ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     4321 caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4381 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4441 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4501 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4561 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4621 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4681 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4741 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4801 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4861 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4921 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     4981 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5041 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5101 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5161 gattttggtc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta
     5221 tgagccatat tcaacgggaa acgtcttgct ctaggccgcg attaaattcc aacatggatg
     5281 ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct
     5341 atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg
     5401 ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc
     5461 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga
     5521 tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg
     5581 ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt
     5641 ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg
     5701 ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag
     5761 aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac
     5821 ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg
     5881 gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc
     5941 cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat
     6001 tgcagtttca tttgatgctc gatgagtttt tctaagaatt aattcatgag cggatacata
     6061 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6121 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     6181 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     6241 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     6301 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     6361 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     6421 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     6481 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     6541 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca
//
