LOCUS       sequence-372791-        7637 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Neq2X DNA polymerase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7637)
  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 7637)
  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-Neq2X
FEATURES             Location/Qualifiers
     source          1..7637
                     /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"
     primer_bind     complement(2949..2967)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2963..3010
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3047..3502
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3134..3153)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3344..3365
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3594..4409)
                     /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(3594..4409)
                     /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     4317..4336
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4401..4420
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4531..5119
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5020..5039
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5273..5290
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5305..5444
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5370..5389)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5530..5552
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5546..5737)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6546..7628)
                     /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     7590..7609
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(7629..7637,1..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 taaataagag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     2941 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     3001 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     3061 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3121 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3181 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3241 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3301 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3361 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3421 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     3481 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3541 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     3601 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     3661 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     3721 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     3781 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     3841 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     3901 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     3961 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     4021 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     4081 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     4141 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     4201 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     4261 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     4321 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     4381 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     4441 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4501 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4561 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4621 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     4681 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4741 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4801 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4861 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4921 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4981 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5041 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5101 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5161 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     5221 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     5281 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     5341 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5401 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     5461 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5521 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5581 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     5641 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     5701 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     5761 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     5821 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     5881 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     5941 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     6001 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     6061 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     6121 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     6181 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     6241 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     6301 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     6361 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     6421 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     6481 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     6541 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6601 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     6661 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     6721 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     6781 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     6841 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     6901 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     6961 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     7021 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     7081 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     7141 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     7201 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     7261 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     7321 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     7381 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     7441 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     7501 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     7561 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     7621 ggtttcacat tcaccac
//
