LOCUS       sequence-117725-        5772 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET-30-based vector dedicated to efficient scFv expression, which 
            circumvents the problem of in-frame amber codons in scFvs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5772)
  AUTHORS   Ossysek K, Uchanski T, Kulesza M, Bzowska M, Klaus T, Wos K, Madej 
            M, Bereta J
  TITLE     A new expression vector facilitating production and functional 
            analysis of scFv antibody fragments selected from Tomlinson I+J 
            phagemid libraries.
  JOURNAL   Immunol Lett. 2015 Jul 25. pii: S0165-2478(15)30011-0. doi: 
            10.1016/j.imlet.2015.07.005.
  PUBMED    26219832
REFERENCE   2  (bases 1 to 5772)
  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: "Immunol 
            Lett. 2015 Jul 25. pii: S0165-2478(15)30011-0. doi: 
            10.1016/j.imlet.2015.07.005."
COMMENT     Sequence Label: pET-scFv-T
FEATURES             Location/Qualifiers
     source          1..5772
                     /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"
     CDS             complement(185..202)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     sig_peptide     complement(632..697)
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     RBS             705..727
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    742..766
                     /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(766..785)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(767..785)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(873..892)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1098..1175
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1176..2258
                     /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(1195..1214)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3067..3258
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3252..3274)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3360..3502
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3415..3434
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3517..3534)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3688..4276)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3768..3787)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4387..4406)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4398..5213
                     /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             4398..5213
                     /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(4471..4490)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5306..5761)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5443..5464)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5655..5674
                     /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 gaaggatccg cgttattaaa
      181 gcttgtgatg gtgatgatga tgtgcggccg cccgtttgat ttccaccttg gtcccttggc
      241 cgaacgtatg aggccgcttc aaaatctgtt gacagtagta agttgcaaaa tcttcaggtt
      301 gcagactgct gatggtgaga gtgaaatctg tcccagatcc actgccactg aaccttgatg
      361 ggaccccact ttgcaacatg gatgcccgat agatcaggag cttaggggct ttccctggtt
      421 tctgctgata ccaatttaaa tagctgctaa tgctctgact tgcccggcaa gtgatggtga
      481 ctctgtctcc tacagatgca gacagggagg atggagactg ggtcatctgg atgtccgtcg
      541 acccgccacc gccgctgcca cctccgcctg aaccgcctcc accgctcgag acggtgacca
      601 gggttccctg gccccagtag tcaaacacct cggccatggc cggctgggcc gcgagtaata
      661 acaatccagc ggctgccgta ggcaataggt atttcatatg tatatctcct tcttaaagtt
      721 aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg
      781 tattaatttc gcgggatcga gatcgatctc gatcctctac gccggacgca tcgtggccgg
      841 catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga
      901 agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg
      961 ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc
     1021 ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg
     1081 agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat
     1141 agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg
     1201 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     1261 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     1321 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     1381 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     1441 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     1501 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     1561 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     1621 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     1681 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     1741 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     1801 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     1861 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     1921 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggacatct
     1981 cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca
     2041 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     2101 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     2161 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     2221 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt
     2281 tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc
     2341 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     2401 atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc
     2461 tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc
     2521 ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt
     2581 atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg
     2641 ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg
     2701 tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt
     2761 ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg
     2821 atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag
     2881 cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt
     2941 ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc
     3001 atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag
     3061 gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag
     3121 acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt
     3181 gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat
     3241 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     3301 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     3361 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat
     3421 cagagcagat tgtactgaga gtgcaccata tatgcggtgt gaaataccgc acagatgcgt
     3481 aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3541 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3601 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3661 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3721 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3781 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3841 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3901 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3961 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4021 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4081 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     4141 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4201 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4261 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4321 cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct tacataaaca
     4381 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct aggccgcgat
     4441 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
     4501 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
     4561 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     4621 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     4681 ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg
     4741 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     4801 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     4861 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     4921 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
     4981 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     5041 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     5101 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     5161 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taagaattaa
     5221 ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5281 acatttcccc gaaaagtgcc acctgaaatt gtaaacgtta atattttgtt aaaattcgcg
     5341 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     5401 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     5461 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     5521 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     5581 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     5641 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     5701 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     5761 tcccattcgc ca
//
