LOCUS       sequence-152171-        6329 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human beta A1-crystallin in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6329)
  TITLE     Larry David lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6329)
  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            "Unpublished"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 CRYBA1
FEATURES             Location/Qualifiers
     source          1..6329
                     /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             1077..1094
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1147..1165)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1161..1208
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1245..1700
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1332..1351)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1542..1563
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1727..1831
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1832..1900,1901..2692)
                     /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(2050..2069)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2863..3451
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3352..3371
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3605..3622
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3637..3779
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3705..3724)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3865..3887
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3881..4072)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4881..5963)
                     /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     5925..5944
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5964..6041)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6247..6266
                     /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 tgctcagacc aaccctacgc cggggtccct ggggccatgg aagataacca
      481 tctatgatca ggagaacttt cagggcaaga ggatggagtt caccagctcc tgtccaaatg
      541 tctctgagcg cagttttgat aatgtccggt ccctgaaggt ggaaagtggc gcctggattg
      601 gttatgagca taccagcttc tgtgggcaac agtttatcct ggagagagga gaataccctc
      661 gctgggatgc ctggagtggg agtaatgcct accacattga gcgtctcatg tccttccgcc
      721 ccatctgttc agctaatcat aaggagtcta agatgaccat ctttgagaag gaaaacttta
      781 ttggacgcca gtgggagatc tctgacgact acccctcctt gcaagccatg ggctggttca
      841 acaacgaagt cggctccatg aagatacaaa gtggggcctg ggtttgctac caatatcctg
      901 gatatcgtgg gtatcagtat atcttggaat gtgaccatca tggaggagac tataaacatt
      961 ggagagagtg gggctctcat gcccagactt cgcagatcca atcgattcgc cgaatccaac
     1021 agtaactaga ggatccgaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc
     1081 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1141 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1201 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     1261 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1321 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1381 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1441 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1501 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     1561 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     1621 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1681 aatattaacg tttacaattt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1741 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     1801 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     1861 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     1921 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     1981 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     2041 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     2101 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     2161 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     2221 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     2281 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     2341 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     2401 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     2461 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     2521 tgataaatct ggagccggtg agcgtggttc tcgcggtatc attgcagcac tggggccaga
     2581 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     2641 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     2701 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     2761 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     2821 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2881 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2941 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3001 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3061 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3121 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3181 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3241 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3301 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3361 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3421 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3481 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3541 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3601 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3661 gcatctgtgc ggtatttcac accgcatata tggtgcactc tcagtacaat ctgctctgat
     3721 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     3781 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3841 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3901 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     3961 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4021 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4081 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4141 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4201 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4261 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4321 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4381 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4441 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4501 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4561 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4621 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4681 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     4741 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     4801 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     4861 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4921 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     4981 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     5041 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5101 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5161 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5221 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5281 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5341 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5401 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5461 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5521 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5581 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5641 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     5701 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     5761 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     5821 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     5881 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     5941 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     6001 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     6061 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     6121 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     6181 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     6241 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     6301 ggccacgatg cgtccggcgt agaggatcg
//
