LOCUS       sequence-152180-        6371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human beta B3-crystallin in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6371)
  TITLE     Larry David lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6371)
  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            "Unpublished"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 CRYBB3
FEATURES             Location/Qualifiers
     source          1..6371
                     /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             1119..1136
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1189..1207)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1203..1250
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1287..1742
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1374..1393)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1584..1605
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1769..1873
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1874..1942,1943..2734)
                     /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(2092..2111)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2905..3493
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3394..3413
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3647..3664
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3679..3821
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3747..3766)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3907..3929
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3923..4114)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4923..6005)
                     /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     5967..5986
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6006..6083)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6289..6308
                     /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 tatggcggaa cagcacggag cacccgaaca ggctgcagct ggcaagagcc
      481 atggagacct tgggggcagc tacaaggtga tcttgtacga actagagaac ttccaaggca
      541 aacgctgcga gctctcggcc gagtgcccca gcctgaccga cagcctgctg gagaaggtgg
      601 gctccatcca agtggagtcc gggccgtggc tggcatttga gtccagggcc ttccgcgggg
      661 agcagtttgt tctggagaag ggggattatc ctcgctggga tgcctggtcc aacagccgtg
      721 atagtgacag ccttctgtcc ctccggcctc tgaatattga tagtccagat cacaagctgc
      781 atctgtttga gaacccagct ttcagtggcc gcaagatgga gatagtggat gatgacgtgc
      841 ccagcctgtg ggctcatggc ttccaggacc gtgtggcgag tgtccgtgcc atcaacggga
      901 cgtgggttgg ctatgagttc cccggctacc gtgggcgcca gtacgtgttt gagcggggcg
      961 agtaccgcca ctggaatgag tgggacgcca gccagccgca gctgcagtct gtgcgccgca
     1021 tccgtgacca gaagtggcac aagcggggcc gcttccccag cagctaacta gaggatccga
     1081 attcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac caccactgag
     1141 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     1201 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     1261 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     1321 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     1381 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     1441 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     1501 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     1561 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     1621 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     1681 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     1741 ttcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1801 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1861 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     1921 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     1981 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     2041 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     2101 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     2161 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     2221 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     2281 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     2341 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     2401 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     2461 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     2521 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     2581 tgagcgtggt tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     2641 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     2701 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     2761 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     2821 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2881 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2941 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3001 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3061 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3121 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3181 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3241 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3301 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3361 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3421 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3481 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3541 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3601 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3661 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3721 acaccgcata tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3781 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     3841 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3901 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3961 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     4021 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     4081 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     4141 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     4201 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4261 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     4321 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4381 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4441 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4501 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4561 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     4621 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4681 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4741 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     4801 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     4861 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     4921 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4981 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     5041 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     5101 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     5161 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     5221 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     5281 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     5341 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5401 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     5461 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5521 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5581 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5641 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5701 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5761 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     5821 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     5881 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     5941 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     6001 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     6061 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     6121 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     6181 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     6241 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     6301 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     6361 gtagaggatc g
//
