LOCUS       sequence-248774-        6064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of IntCtr-GyrA fusion protein (with N- and 
            C-terminal His6).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6064)
  TITLE     Muir Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6064)
  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            "Unpublished"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: pTXB1-His6-IntCtr-GyrA-His6
FEATURES             Location/Qualifiers
     source          1..6064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     33..52
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        33..51
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    52..76
                     /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             91..113
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             241..834
                     /codon_start=1
                     /gene="Mycobacterium xenopi gyrA"
                     /product="modified GyrA intein (Southworth et al., 1999)"
                     /label=Mxe GyrA intein
                     /note="N-terminal cleavage of this intein can be induced by
                     thiols."
                     /translation="CITGDALVALPEGESVRIADIVPGVRPNSDNAIDLKVLDRHGNPV
                     LADRLFHSGEHPVYTVRTVEGLRVTGTANHRLLCLVDVAGVPTLLWKLIDEIKPGDYAV
                     IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY
                     AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA"
     CDS             835..852
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             886..1041
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     primer_bind     complement(1106..1124)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1120..1166
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1227..1331
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1332..1400,1401..2192)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1550..1569)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(2234..2747)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2429..2450)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2641..2660
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      2858..3446
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3347..3366
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3600..3617
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3639..3658)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3799..3821
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3815..4006)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4563..5645)
                     /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     5607..5626
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5646..5723)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      5876..5962
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ggtttaaacc ggggatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
       61 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
      121 atggctcatc accaccatca tcacggcgtc aagatcatta gtcgtaagag tctgggcact
      181 caaaatgtct acgatattgg agtagaaaaa gatcataatt ttttggcggc tactgggggt
      241 tgcatcacgg gagatgcact agttgcccta cccgagggcg agtcggtacg catcgccgac
      301 atcgtgccgg gtgtgcggcc caacagtgac aacgccatcg acctgaaagt ccttgaccgt
      361 catggcaatc ccgtgctcgc cgaccggctg ttccactccg gcgagcatcc ggtgtacacg
      421 gtgcgtacgg tcgaaggtct gcgtgtgacg ggcaccgcga accaccggtt gttgtgtttg
      481 gtcgacgtcg ccggggtgcc gaccctgctg tggaagctga tcgacgaaat caagccgggc
      541 gattacgcgg tgattcaacg cagcgcattc agcgtcgact gtgcaggttt tgcccgcggg
      601 aaacccgaat ttgcgcccac aacctacaca gtcggcgtcc ctggactggt gcgtttcttg
      661 gaagcacacc accgagaccc ggacgcccaa gctatcgccg acgagctgac cgacgggcgg
      721 ttctactacg cgaaagtcgc cagtgtcacc gacgccggcg tgcagccggt gtatagcctt
      781 cgtgtcgaca cggcagacca cgcgtttatc acgaacgggt tcgtcagcca cgctcatcat
      841 catcatcatc attaaactgg cctcaccggt ctgaactcag gcctcacgac aaatcctggt
      901 gtatccgctt ggcaggtcaa cacagcttat actgcgggac aattggtcac atataacggc
      961 aagacgtata aatgtttgca gccccacacc tccttggcag gatgggaacc atccaacgtt
     1021 cctgccttgt ggcagcttca atgactgcag gaaggggatc cggctgctaa caaagcccga
     1081 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1141 tctaaacggg tcttgagggg ttttttactg aaaggaggaa ctatatccgg ataactacgt
     1201 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1261 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1321 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1381 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     1441 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     1501 gttttcgccc cgaagaacgt ttcccaatga tgagcacttt taaagttctg ctatgtggcg
     1561 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     1621 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     1681 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1741 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1801 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     1861 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     1921 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     1981 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2041 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2101 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2161 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     2221 tttagattga tttaccccgg ttgataatca gaaaagcccc aaaaacagga agattgtata
     2281 agcaaatatt taaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     2341 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2401 atagcccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2461 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2521 accatcaccc aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2581 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2641 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2701 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtaaa aggatctagg
     2761 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2821 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2881 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2941 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3001 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3061 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3121 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3181 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3241 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3301 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3361 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3421 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3481 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3541 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3601 gcgagtcagt gagcgaggaa gctatggtgc actctcagta caatctgctc tgatgccgca
     3661 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3721 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3781 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3841 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgcagcgat tcacagatgt
     3901 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     3961 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4021 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4081 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4141 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag ccgaacgcca
     4201 gcaagacgta gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga
     4261 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4321 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4381 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     4441 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     4501 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     4561 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4621 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     4681 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     4741 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     4801 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     4861 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     4921 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     4981 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5041 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     5101 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5161 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5221 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5281 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5341 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5401 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     5461 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     5521 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     5581 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     5641 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     5701 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     5761 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     5821 atgccggcat gccgcccttt cgtcttcaag aattaattcc caattcccca ggcatcaaat
     5881 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     5941 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
     6001 cggagggtgg cgggcaggac gcccgccata aactgccagg aattggggat cggaattaat
     6061 tccc
//
