LOCUS       sequence-233162-        6273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pNIC expression vector encoding gene for YopH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6273)
  TITLE     YopH-pNIC-Bsa4
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6273)
  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            "Unpublished"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: YopH-pNIC-Bsa4
FEATURES             Location/Qualifiers
     source          1..6273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1138..1155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1208..1226)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1222..1269
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1306..1761
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1393..1412)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1603..1624
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1853..2668)
                     /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(1853..2668)
                     /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     2576..2595
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2660..2679
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2790..3378
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3279..3298
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3532..3549
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3564..3703
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3629..3648)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3789..3811
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3805..3996)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4805..5887)
                     /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     5849..5868
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5888..5965)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6171..6190
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatgcgt gaacgtcctc acaccagcgg
      181 tcaccacggt gcgggtgagg cgcgtgcgac ggctccttct accgtttctc catacggtcc
      241 ggaagcccgt gcggaactga gctctcgtct gaccaccctg cgtaataccc tggctccggc
      301 gaccaacgac ccgcgttacc tccaggcgtg cggtggtgaa aaactgaacc gttttcgtga
      361 catccagtgt cgtcgccaga ctgctgtacg cgcggacctg aacgcgaact atatccaggt
      421 cggtaacacc cgtaccatcg cgtgccagta tccgctgcaa tctcaactgg agtctcactt
      481 ccgtatgctg gccgaaaacc gcaccccggt gctcgcggtt ctggcgtcct cctctgaaat
      541 cgctaaccag cgctttggta tgccggacta ctttcgtcaa tctggtacct acggctctat
      601 caccgttgaa tctaagatga cccagcaagt tggcctgggt gacggtatca tggcggacat
      661 gtacacgctg accatccgtg aagccggtca gaaaaccatc agcgtcccag tggtccacgt
      721 tggtaactgg ccggatcaga ccgcagttag cagcgaggtt acgaaagccc tcgcgtctct
      781 ggttgaccaa acggcggaga ctaaacgtaa catgtatgaa agcaaaggtt cttctgcggt
      841 tgcggacgac tctaaactgc gtcctgttat tcactgccgt gccggtgtag gtcgtaccgc
      901 gcagctgatc ggtgcgatgt gcatgaacga ttctcgtaac agccagctgt ctgtcgaaga
      961 tatggtttct cagatgcgtg ttcagcgtaa tggcatcatg gttcagaaag acgaacaact
     1021 cgacgtactc atcaaactgg cggaaggtca gggtcgtcca ctgctgaact cttaacagta
     1081 aaggtggata cggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac
     1141 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     1201 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1261 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     1321 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     1381 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     1441 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     1501 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1561 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1621 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1681 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     1741 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1801 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     1861 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     1921 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     1981 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     2041 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     2101 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     2161 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     2221 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     2281 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     2341 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     2401 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     2461 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     2521 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     2581 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     2641 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     2701 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2761 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2821 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2881 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     2941 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3001 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3061 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3121 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3181 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3241 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3301 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3361 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3421 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3481 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3541 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3601 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3661 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     3721 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3781 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3841 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     3901 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     3961 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     4021 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     4081 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     4141 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     4201 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     4261 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     4321 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     4381 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     4441 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     4501 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     4561 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     4621 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     4681 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     4741 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     4801 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4861 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     4921 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     4981 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5041 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     5101 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5161 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5221 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5281 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5341 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5401 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5461 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5521 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5581 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5641 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     5701 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     5761 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     5821 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     5881 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     5941 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6001 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6061 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6121 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     6181 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     6241 gcgtagagga tcgagatctc gatcccgcga aat
//
