LOCUS       sequence-153519-        6261 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human gamma B-crystallin in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6261)
  TITLE     Larry David lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6261)
  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            "Unpublished"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: pE-SUMO CRYGB
FEATURES             Location/Qualifiers
     source          1..6261
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    40..64
                     /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             79..101
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             114..131
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             144..431
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated and modified)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /note="modified to prevent cleavage by eukaryotic SUMO 
                     proteases"
                     /translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR
                     LMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
     CDS             1009..1026
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1079..1097)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1093..1140
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1177..1632
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1264..1283)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1474..1495
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1659..1763
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1764..1832,1833..2624)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1982..2001)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2795..3383
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3284..3303
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3537..3554
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3569..3711
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3637..3656)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3797..3819
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3813..4004)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4813..5895)
                     /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     5857..5876
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5896..5973)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6179..6198
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
       61 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggtcatcacc
      121 atcatcatca cgggtccctg caggactcag aagtcaatca agaagctaag ccagaggtca
      181 agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga tcttcagaga
      241 tcttcttcaa gatcaaaaag accactcctt taagaaggct gatggaagcg ttcgctaaaa
      301 gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga attcaagctg
      361 atcaggcccc tgaagatttg gacatggagg ataacgatat tattgaggct caccgcgaac
      421 agattggagg tggaaagatc accttctacg aggacagggc cttccagggc cgcagctacg
      481 aatgcaccac tgactgcccc aacctacaac cctatttcag ccgctgcaac tccatcaggg
      541 tggagagcgg ctgctggatg atctatgagc gccccaacta ccagggccac cagtacttcc
      601 tgcggcgtgg ggagtacccc gactaccagc aatggatggg cctcagcgac tccatccgct
      661 cctgctgcct catccccccg cactctggcg cttacagaat gaagatctac gacagagatg
      721 aattgagggg acaaatgtca gagctcacag acgactgtct ctctgttcag gaccgcttcc
      781 acctcactga aattcactcc ctcaatgtgc tggagggcag ctggatcctc tatgagatgc
      841 ccaactacag ggggaggcag tatctgctga ggccggggga gtacaggagg tttcttgatt
      901 ggggggctcc aaatgccaaa gttggctctc ttagacgagt catggatttg tactaatcta
      961 gaggatccga attcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac
     1021 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1081 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1141 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     1201 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1261 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1321 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1381 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1441 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1501 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1561 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1621 cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1681 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     1741 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     1801 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     1861 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     1921 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     1981 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     2041 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2101 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     2161 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     2221 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     2281 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     2341 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     2401 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     2461 ctggagccgg tgagcgtggt tctcgcggta tcattgcagc actggggcca gatggtaagc
     2521 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     2581 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     2641 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     2701 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     2761 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     2821 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     2881 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     2941 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3001 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3061 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3121 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3181 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3241 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3301 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3361 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3421 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3481 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3541 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3601 gcggtatttc acaccgcata tatggtgcac tctcagtaca atctgctctg atgccgcata
     3661 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3721 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3781 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3841 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     3901 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     3961 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     4021 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     4081 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     4141 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     4201 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     4261 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     4321 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     4381 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     4441 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     4501 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     4561 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     4621 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     4681 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     4741 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     4801 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4861 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     4921 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     4981 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     5041 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     5101 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     5161 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     5221 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     5281 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     5341 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     5401 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     5461 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     5521 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     5581 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     5641 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     5701 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     5761 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     5821 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     5881 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     5941 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     6001 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     6061 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     6121 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     6181 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     6241 tgcgtccggc gtagaggatc g
//
