LOCUS       sequence-153525-        6330 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 6330)
  TITLE     Larry David lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6330)
  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            "Unpublished"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..6330
                     /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             1078..1095
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1148..1166)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1162..1209
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1246..1701
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1333..1352)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1543..1564
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1728..1832
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1833..1901,1902..2693)
                     /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(2051..2070)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2864..3452
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3353..3372
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3606..3623
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3638..3780
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3706..3725)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3866..3888
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3882..4073)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4882..5964)
                     /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     5926..5945
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5965..6042)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6248..6267
                     /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 agtaatctag aggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac
     1081 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     1141 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1201 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     1261 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     1321 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     1381 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     1441 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1501 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1561 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1621 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     1681 aaatattaac gtttacaatt tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1741 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1801 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1861 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1921 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1981 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2041 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     2101 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2161 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2221 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2281 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2341 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     2401 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2461 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2521 ctgataaatc tggagccggt gagcgtggtt ctcgcggtat cattgcagca ctggggccag
     2581 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2641 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2701 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2761 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2821 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2881 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2941 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3001 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3061 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3121 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3181 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3241 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3301 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3361 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3421 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3481 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3541 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3601 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3661 cgcatctgtg cggtatttca caccgcatat atggtgcact ctcagtacaa tctgctctga
     3721 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3781 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3841 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3901 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     3961 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     4021 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     4081 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     4141 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     4201 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     4261 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     4321 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     4381 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     4441 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     4501 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     4561 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4621 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     4681 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     4741 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     4801 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     4861 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4921 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4981 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     5041 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     5101 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     5161 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5221 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5281 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5341 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5401 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5461 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5521 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5581 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5641 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5701 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5761 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5821 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5881 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5941 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     6001 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     6061 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     6121 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6181 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     6241 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     6301 cggccacgat gcgtccggcg tagaggatcg
//
