LOCUS       sequence-372790-        7869 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Neq2X7 DNA polymerase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7869)
  AUTHORS   Hernandez-Rollan C, Ehrmann AK, Vlassis A, Kandasamy V, Norholm MHH
  TITLE     Neq2X7: a multi-purpose and open-source fusion DNA polymerase for 
            advanced DNA engineering and diagnostics PCR.
  JOURNAL   BMC Biotechnol. 2024 Apr 2;24(1):17. doi: 
            10.1186/s12896-024-00844-7.
  PUBMED    38566117
REFERENCE   2  (bases 1 to 7869)
  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: "BMC 
            Biotechnol."; date: "2024-04-2"; volume: "24(1):17. doi"; pages: " 
            10.1186/s12896-024-00844-7"
COMMENT     Sequence Label: pET-Neq2X7
FEATURES             Location/Qualifiers
     source          1..7869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     275..294
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     378..397
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        378..396
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    397..421
                     /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             436..458
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             468..485
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3111..3128
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3181..3199)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3195..3242
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3279..3734
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3366..3385)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3576..3597
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3826..4641)
                     /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             complement(3826..4641)
                     /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     4549..4568
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4633..4652
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4763..5351
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5252..5271
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5505..5522
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5537..5676
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5602..5621)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5762..5784
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5778..5969)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6778..7860)
                     /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     7822..7841
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(7861..7869,1..69))
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
       61 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
      121 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
      181 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
      241 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
      301 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
      361 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
      421 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgcat caccatcacc
      481 atcacggatc aatgttacac caactcccca cgatggttgt agaagaaaag gcggtaaaag
      541 aggaagaagg gtatagcgtg ctaaaatgtt attggattaa tatagagaac acccctttag
      601 acgaggtaat tttaataggt aaagacgaaa ataatagagc ttgtgaagtt ataattccat
      661 acaaatggta tttctatttt gaaggcgata taaaggattt agaagaattc gctaacaaca
      721 aaaaaataaa aatcgaatat acaaaggagc aaaagaaata tatagaaaaa ccaaaagatg
      781 tttataaagt atatgttttg cataaacatt atccaatact aaaagaattc attaaagaaa
      841 agggctataa aaaatacgaa accgatataa atgtttatag gaagttttta atagataaag
      901 ggatagagcc ttttgaatgg tttgaggtag aaggcaaaat tttattatct acctctaaca
      961 aagttagaat aaaagcacaa agtataaaaa gattgtatga aaagactaag ccatcggttt
     1021 tagcttttga tatagaagtt tacagtgagg ctttccctaa tcctgaaaaa gacaaaataa
     1081 tatctatagc cctttatgga gacaattacg aaggggttat ctcttacaaa ggagaaccaa
     1141 ctataaaagt taataccgaa tatgaattaa ttgagaaatt tgtcgaaata atagaaagct
     1201 taaaaccaga cataatagtt acatacaatg gggataattt cgatatagac tttttagtga
     1261 aaagggcttc tttatacaat ataaggctac caataaaatt ggttaacaaa aaagagccta
     1321 cttataattt tagggaaagc gcacatgtag atttgtataa aacaattact accatatata
     1381 aaacccaatt gtctacccaa acatattcat taaatgaagt agctaaagaa attcttggag
     1441 aggagaaaat ttatgattat gaaaacatgt tatatgattg ggccataggc aattataaca
     1501 aagtgttcga atacaattta aaagatgccg aattaacata taagctattc aaatactatg
     1561 aaaatgattt attggaatta gcaagattgg ttaaccaacc attatttgat gtatctaggt
     1621 ttagctatag taatatagtt gaatggtatc taatcaaaaa aagcagaaaa tataatgaaa
     1681 ttgtgcctaa caaaccaaaa atggaagaag tagagagaag aaaattaaat acctatgcag
     1741 gagcattcgt ttacgaacca aaacccggtt tgtatgagaa tttagctgta ctggatttcg
     1801 cttctctgta tccttcaatt atattagagc ataatgtttc tccaggcaca atatattgtg
     1861 agcatgatga ttgtaaacaa aatggggtag aagcgataat aaataatgag aaaaaatatg
     1921 tgtggttttg caaaaaagta aaagggttta ttccaacggt attagagcat ttgtatacaa
     1981 aaaggctaga acttaagaga aaactgaaag aactagatag ggatagtgaa gaatataaaa
     2041 ttataaatgc taagcaaaga gtattgaaaa taataattaa tgcaacctat ggctatatgg
     2101 gtttcccaag agcgagatgg tattgcatag actgtgctgc ggcagtagca gcttggggca
     2161 ggaaatacat taattatata ttaaaaaggg ccgaagaaga aggattcaaa gtaatttatg
     2221 gagataccga ttcattattc atttctgggg acaaagacaa agtattagaa tttttagaga
     2281 aagtaaataa agaattaccc ggtaaaatac aattagattt agaagatttc tatgttagag
     2341 ggatattcgt aaaaaagagg ggtgaacaaa agggggcaaa aaagaaatat gctttattaa
     2401 gcgaacaagg ttacataaag ctaaggggct tcgaagcagt aagaacagac tgggctccca
     2461 tagttaaaga agtccaaaca aagctattgg aaattttgct aaaagaaggt aacatagaaa
     2521 aagcaagaca atacataaaa gaaattatta gaaagctaag aaatagagaa ataccatggg
     2581 agaagctttt aattacagaa acgataagaa agcctttaga aaaatacaaa gttgaagctc
     2641 ctcatgtggc agcagcaaaa aaatataaaa ggttgggcta taaagttatg cctggcttta
     2701 gagttagata tttagtggta ggtagcactg gaagggtttc agatagaatt aaaatagaca
     2761 aagaagttag gggtaatgaa tatgaccccg aatactacat agaaaaacaa ctattgcctg
     2821 cagtagagca aatattagaa tctgtaggta ttaaagacac attcacaggc aaaaaactaa
     2881 cagatttctt taaaggtacc ggcggtggcg gtgcaaccgt aaagttcaag tacaaaggcg
     2941 aagaaaaaga ggtagacatc tccaagatca agaaagtatg gcgtgtgggc aagatgatct
     3001 ccttcaccta cgacgagggc ggtggcaaga ccggccgtgg tgcggtaagc gaaaaggacg
     3061 cgccgaagga gctgctgcag atgctggaga agcagaaaaa gtagctcgag caccaccacc
     3121 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     3181 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     3241 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     3301 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3361 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3421 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3481 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3541 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3601 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3661 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     3721 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3781 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     3841 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     3901 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     3961 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     4021 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     4081 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     4141 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     4201 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     4261 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     4321 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     4381 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     4441 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     4501 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     4561 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     4621 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     4681 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4741 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4801 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4861 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     4921 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4981 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5041 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5101 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5161 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5221 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5281 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5341 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5401 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     5461 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     5521 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     5581 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     5641 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     5701 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5761 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     5821 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     5881 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     5941 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     6001 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     6061 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     6121 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     6181 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     6241 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     6301 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     6361 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     6421 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     6481 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     6541 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     6601 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     6661 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     6721 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     6781 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6841 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     6901 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     6961 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     7021 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     7081 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     7141 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     7201 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     7261 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     7321 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     7381 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     7441 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     7501 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     7561 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     7621 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     7681 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     7741 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     7801 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     7861 attcaccac
//
