LOCUS       sequence-466060-        5524 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for 10xHis-phi29 DNA polymerase (catalytically 
            inactive mutant D249E): PT7-lacO-g10leader-p2 dead-T7 terminator, 
            CamR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5524)
  AUTHORS   Barthel S, Hoffmann-Becking M, Karimov IG, Erb TJ
  TITLE     Genetically encoded control of in vitro transcription-translation 
            coupled DNA replication
  JOURNAL   bioRxiv 2025.07.08.663768; doi: 10.1101/2025.07.08.663768
REFERENCE   2  (bases 1 to 5524)
  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: "bioRxiv 
            2025.07.08.663768; doi: 10.1101/2025.07.08.663768"
COMMENT     Sequence Label: pTE5425
FEATURES             Location/Qualifiers
     source          1..5524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    973..994
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     terminator      997..1040
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     complement(1044..1062)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             1224..1883
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1290..1309)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1904..1998
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      2181..2769
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2670..2689
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2923..2940
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2964..3021)
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     CDS             complement(4794..4805)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(4818..4847)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             4860..4882
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4897..4921
                     /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(4921..4940)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4922..4940)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5024..5043)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5249..5326
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(5346..5365)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
       61 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
      121 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
      181 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
      241 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
      301 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
      361 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
      421 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
      481 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
      541 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
      601 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
      661 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
      721 ctctctcagg gccaggcggt gaagggcaat cagctgttgc cagtctcact ggtgaaaaga
      781 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
      841 atgcagctgg cacgacagtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
      901 gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac
      961 ccagactcat cgctctagaa gcggccgcga attccagaaa tcatccttag cgaaagctaa
     1021 ggattttttt tatctgaaat tctgcctcgt gatacgccta tttttatagg ttaatgtcat
     1081 gataataatg gtttcttaga cgtcaggtgg cactcgagtt gatcgggcac gtaagaggtt
     1141 ccaactttca ccataatgaa ataagatcac taccgggcgt attttttgag ttatcgagat
     1201 tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata
     1261 tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct
     1321 ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc
     1381 acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat
     1441 ttcgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca
     1501 ccgttttcca tgagcaaact gaaacgtttt catcactctg gagtgaatac cacgacgatt
     1561 tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct
     1621 atttccctaa agggtttatt gagaatatgt ttttcgtatc agccaatccc tgggtgagtt
     1681 tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca
     1741 tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc
     1801 atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg
     1861 acgagtggca gggcggggcg taatttgata tcgagctcgc ttggactcct gttgatagat
     1921 ccagtaatga cctcagaact ccatctggat ttgttcagaa cgctcggttg ccgccgggcg
     1981 ttttttattg gtgagaatcc aagcctcgag ctgtcagacc aagtttactc atatatactt
     2041 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2101 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2161 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2221 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2281 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     2341 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2401 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2461 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2521 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2581 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2641 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2701 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     2761 ctgtggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     2821 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     2881 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     2941 gaagcctgca taacgcgaag taatcttttc ggttttaaag aaaaagggca gggtggtgac
     3001 accttgccct tttttgccgg actgcagcgg ccgctactag tcgcgatgag tggtctcaaa
     3061 gcttatttga tcgtgaaggt gtcatcgacc agcacaacgc cgcccggaac ttggaccggt
     3121 ttcggtttca ttttgcgact gaaacccact ttgaaatttt cgaacgtcac ttctttctta
     3181 attttatcgg tcatgccggc gcatttcacc gaaaatttaa tgtcggtata gtcatccggt
     3241 gaaccttcca ccagtttgcc atcgacttct ttcatgtaga tgtcttggat gtacgttttc
     3301 tggcgcaggt atttagcacg tttaaaggtc gattcgtgtg cccaataacc cagttttttc
     3361 gggtcaacga tatctttaat cacatccggg atttccgtgc cggtcagatg aattgagtcg
     3421 gtatcacagt agataatgcg gtcatagcac gcttgtgcgg cggtgatggt ggtgtaacgt
     3481 gcccacgcgg taatgaaaac acccatcggc gtatacaccg gatctttggt ttcttcttcg
     3541 cccaggcgaa aacccagagc gccattttct ttcaggtacg ggactttacc ggtaacatcc
     3601 ggattagatg caaatttgcc atacaggctg ttcagcatca gtttagccag ctgtttgatt
     3661 gcaccttcag acgtggtttt gatgtaggtc catttgtcaa tgaaatcttt aaacagaccc
     3721 gtggtggctt taaatttcag gccggagatg tattcaacgt tgtacagatc gtaatgttct
     3781 ttcatcagtt ccagatcgac gttactcagc cacaggtctg cgatttcacc gccagagctt
     3841 ttcaggtatt cgttaccctt atagaagcgg ctacgtttga tttgaatggt cggaatatag
     3901 ccttctttca gttcaaattc acaacggatg tgctgaatat gcagcgggta gtcttcatcc
     3961 cagacatatt taccctcgaa aacgatcggt tcgccatacg gcagcaggcg ggagtacatc
     4021 tgagccggat acagactatt cacttcaaag accataccct cgccaatttc tttttctttg
     4081 aaacggtcgt tcagccacgt aaaaccgccg cggtatgcat aacgaacttc tttatccaga
     4141 cccaggctca gggtcgggaa cactttctta aattttttcg tggtgatgat atctttgaaa
     4201 cctttcaggg agtcactgcc ggcggtcatg cgatccaggc cttgtttaaa ctgaatcagc
     4261 agagcttctg cgataatctg gatatcgttt ttaatgtacg catattcttc cggcgtgatt
     4321 ttgtaaccaa ccggacgttc tttgtgatag tcaatatcgc ctttcaggac ggtcagtttg
     4381 aaatctttcg caattttctt caccggaaac ggcagttttt tcaggctgtc atagatcacg
     4441 gtatgaattt tgcgtttacc tttgtagccc aggcagatat caatcatata ccattggccc
     4501 atacgagaga taatcgtatt ataggtgttc ggcagaccat cggcgctcca tttaaagcca
     4561 ttacgttcca gccagttgat aatgaacgca ccatcaaatt tcagattgtg gaagtacagg
     4621 tcggcctgaa ctttcagcac ccacgccata aattcatcca gcgagttgcc aattttatat
     4681 tctgaatggt cttcgatgtt catataacca taggcccaga cacggcaatc ttcaactttc
     4741 gtcgtcgttt caaaatcgca gctgtacatt ttacgcggca ttcgagaccc atgacgacct
     4801 tcgatatggc cgctgctgtg atgatgatga tgatgatgat gatgatggcc catggtatat
     4861 ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc
     4921 ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta cgccggacgc
     4981 atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc
     5041 accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt
     5101 atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc
     5161 cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag
     5221 tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg
     5281 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
     5341 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     5401 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     5461 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     5521 cgtt
//
