LOCUS       sequence-462633-        6408 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Francisella tularensis Histidine Acid Phosphatase under T7
            with HIS tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6408)
  AUTHORS   Beckham J
  TITLE     Josh Beckham Lab plasmids 2024
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6408)
  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            "Unpublished"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: FtHAP-pNIC-Bsa4
FEATURES             Location/Qualifiers
     source          1..6408
                     /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             1273..1290
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1343..1361)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1357..1404
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1441..1896
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1528..1547)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1738..1759
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1988..2803)
                     /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(1988..2803)
                     /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     2711..2730
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2795..2814
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2925..3513
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3414..3433
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3667..3684
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3699..3838
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3764..3783)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3924..3946
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3940..4131)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4940..6022)
                     /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     5984..6003
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6023..6100)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6306..6325
                     /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 atccatgaag aaaatctttg ttagctttac
      181 tctgctcttc ttcctgattc cggtgggtta ctcttctaag ctgatcttcg tgtctatgat
      241 cacccgtcac ggcgatcgtg cgccgtttgc gaacatcgaa aacgcgaact actcttgggg
      301 taccgaactg tctgagctga ccccaatcgg tatgaaccag gaatacaatc tgggtctcca
      361 gctgcgtaaa cgttacatcg acaaattcgg tctgctgccg gagcactacg ttgaccaatc
      421 tatctacgtc ctgtcttctc acacgaatcg taccgttgtt tctgcgcaat ctctgctgat
      481 gggtctgtac ccagcgggta cgggtccgct gatcggtgac ggcgacccgg caatcaaaga
      541 ccgtttccag ccgatcccga tcatgaccct gtctgcggac tctcgtctga tccagttccc
      601 gtacgaacag tacctggcgg ttctgaaaaa gtacgtgtac aacagcccgg agtggcaaaa
      661 caaaaccaaa gaagcggctc cgaactttgc caaatggcag caaatcctgg gtaatcgtat
      721 ctctggtctg aacgacgtca tcaccgttgg tgacgttctg atcgtggccc aggcgcatgg
      781 caagccgctg ccgaaaggtc tgtctcaaga agacgcggac cagatcatcg cgctgaccga
      841 ctggggtctg gcgcagcagt tcaaatctca gaaagtttct tacatcatgg gtggtaaact
      901 gaccaaccgt atgatcgaag acctcaacaa cgcggttaat ggtaaatcta aatacaaaat
      961 gacctactac tccggtcacg acctcaccct gctcgaagtt atgggtactc tgggtgttcc
     1021 gctggacacc gccccaggtt acgcgagcaa cctggaaatg gaactgtaca aggatggtga
     1081 catctacacc gttaaactgc gttacaacgg caagtatgtg aaactgccta tcatggacaa
     1141 aaacaactct tgctctctgg acgctctgaa caaatacatg cagtctatca acgaaaaatt
     1201 ccagaaataa cagtaaaggt ggatacggat ccgaattcga gctccgtcga caagcttgcg
     1261 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     1321 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     1381 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     1441 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1501 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1561 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1621 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1681 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1741 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1801 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1861 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     1921 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     1981 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     2041 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     2101 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     2161 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     2221 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     2281 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     2341 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     2401 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     2461 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     2521 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     2581 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     2641 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     2701 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     2761 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     2821 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     2881 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2941 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3001 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3061 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3121 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3181 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3241 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3301 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3361 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3421 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3481 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3541 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3601 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3661 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3721 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     3781 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     3841 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     3901 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     3961 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     4021 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     4081 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     4141 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     4201 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4261 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4321 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     4381 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     4441 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     4501 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     4561 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     4621 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     4681 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     4741 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     4801 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     4861 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     4921 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4981 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     5041 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     5101 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     5161 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     5221 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5281 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5341 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5401 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     5461 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5521 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     5581 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     5641 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     5701 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     5761 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     5821 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     5881 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     5941 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     6001 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     6061 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     6121 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     6181 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6241 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6301 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     6361 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaat
//
