LOCUS       sequence-78800-e        7370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of superfolder GFP for protein purification 
            (Intein-chiting binding domain fussion, New England Biolabs pTXB1 
            backbone).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7370)
  TITLE     Plamids for protein purification in E. coli of superfolder GFP, 
            CRP/CAP (E. coli), FIS (E. coli), CI (lambda phage) and CII (lambda 
            phage)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7370)
  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            "Unpublished"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-sfGFP
FEATURES             Location/Qualifiers
     source          1..7370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..139
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(140..208,209..1000)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(358..377)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1042..1555)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1237..1258)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1449..1468
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      1666..2254
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2155..2174
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2408..2425
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2447..2466)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2607..2629
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2623..2814)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3371..4453)
                     /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     4415..4434
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4454..4531)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      4684..4770
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4867..4953
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5050..5136
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5233..5319
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5416..5502
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5637..5656
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5637..5655
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5656..5680
                     /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             5695..5717
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5726..6439
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             6440..7033
                     /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="CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPV
                     LADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAV
                     IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY
                     AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA"
     CDS             7064..7219
                     /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(7284..7302)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7298..7345
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 aactacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
       61 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
      121 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
      181 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
      241 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
      301 ccttgagagt tttcgccccg aagaacgttc tccaatgatg agcactttta aagttctgct
      361 atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca
      421 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
      481 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
      541 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
      601 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
      661 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
      721 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
      781 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
      841 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
      901 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
      961 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     1021 atatatactt tagattgatt taccccggtt gataatcaga aaagccccaa aaacaggaag
     1081 attgtataag caaatattta aattgtaaac gttaatattt tgttaaaatt cgcgttaaat
     1141 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     1201 tcaaaagaat agcccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     1261 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     1321 ctacgtgaac catcacccaa atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     1381 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     1441 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     1501 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtaaaag
     1561 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     1621 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     1681 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     1741 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     1801 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     1861 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     1921 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     1981 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2041 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2101 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2161 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2221 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2281 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     2341 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     2401 cgagcgcagc gagtcagtga gcgaggaagc tatggtgcac tctcagtaca atctgctctg
     2461 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     2521 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     2581 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     2641 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gcagcgattc
     2701 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     2761 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     2821 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     2881 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     2941 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcc
     3001 gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt
     3061 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     3121 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     3181 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     3241 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     3301 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     3361 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3421 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     3481 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     3541 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     3601 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     3661 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     3721 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     3781 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     3841 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     3901 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     3961 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     4021 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     4081 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     4141 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     4201 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     4261 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     4321 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     4381 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     4441 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     4501 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     4561 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     4621 aatggtgcat gccggcatgc cgccctttcg tcttcaagaa ttaattccca attccccagg
     4681 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     4741 tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag
     4801 caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggaa ttaattcccc
     4861 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     4921 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
     4981 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gaattaattc
     5041 cccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     5101 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt
     5161 gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggaattaa
     5221 ttccccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     5281 tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac
     5341 gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggaat
     5401 taattcccca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     5461 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg
     5521 aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg
     5581 aattggggat cggaattaat tcccggttta aaccggggat ctcgatcccg cgaaattaat
     5641 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     5701 taactttaag aaggagatat acataatgcg taaaggcgaa gagctgttca ctggtgtcgt
     5761 ccctattctg gtggaactgg atggtgatgt caacggtcat aagttttccg tgcgtggcga
     5821 gggtgaaggt gacgcaacta atggtaaact gacgctgaag ttcatctgta ctactggtaa
     5881 actgccggta ccttggccga ctctggtaac gacgctgact tatggtgttc agtgctttgc
     5941 tcgttatccg gaccatatga agcagcatga cttcttcaag tccgccatgc cggaaggcta
     6001 tgtgcaggaa cgcacgattt cctttaagga tgacggcacg tacaaaacgc gtgcggaagt
     6061 gaaatttgaa ggcgataccc tggtaaaccg cattgagctg aaaggcattg actttaaaga
     6121 agacggcaat atcctgggcc ataagctgga atacaatttt aacagccaca atgtttacat
     6181 caccgccgat aaacaaaaaa atggcattaa agcgaatttt aaaattcgcc acaacgtgga
     6241 ggatggcagc gtgcagctgg ctgatcacta ccagcaaaac actccaatcg gtgatggtcc
     6301 tgttctgctg ccagacaatc actatctgag cacgcaaagc gttctgtcta aagatccgaa
     6361 cgagaaacgc gatcatatgg ttctgctgga gttcgtaacc gcagcgggca tcacgcatgg
     6421 tatggatgaa ctgtacaaat gcatcacggg agatgcacta gttgccctac ccgagggcga
     6481 gtcggtacgc atcgccgaca tcgtgccggg tgcgcggccc aacagtgaca acgccatcga
     6541 cctgaaagtc cttgaccggc atggcaatcc cgtgctcgcc gaccggctgt tccactccgg
     6601 cgagcatccg gtgtacacgg tgcgtacggt cgaaggtctg cgtgtgacgg gcaccgcgaa
     6661 ccacccgttg ttgtgtttgg tcgacgtcgc cggggtgccg accctgctgt ggaagctgat
     6721 cgacgaaatc aagccgggcg attacgcggt gattcaacgc agcgcattca gcgtcgactg
     6781 tgcaggtttt gcccgcggga aacccgaatt tgcgcccaca acctacacag tcggcgtccc
     6841 tggactggtg cgtttcttgg aagcacacca ccgagacccg gacgcccaag ctatcgccga
     6901 cgagctgacc gacgggcggt tctactacgc gaaagtcgcc agtgtcaccg acgccggcgt
     6961 gcagccggtg tatagccttc gtgtcgacac ggcagaccac gcgtttatca cgaacgggtt
     7021 cgtcagccac gctactggcc tcaccggtct gaactcaggc ctcacgacaa atcctggtgt
     7081 atccgcttgg caggtcaaca cagcttatac tgcgggacaa ttggtcacat ataacggcaa
     7141 gacgtataaa tgtttgcagc cccacacctc cttggcagga tgggaaccat ccaacgttcc
     7201 tgccttgtgg cagcttcaat gactgcagga aggggatccg gctgctaaca aagcccgaaa
     7261 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     7321 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
//
