LOCUS       sequence-153503-        6384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human beta A3-crystallin in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6384)
  TITLE     Larry David lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6384)
  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            "Unpublished"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 CRYBA3
FEATURES             Location/Qualifiers
     source          1..6384
                     /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             1132..1149
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1202..1220)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1216..1263
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1300..1755
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1387..1406)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1597..1618
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1782..1886
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1887..1955,1956..2747)
                     /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(2105..2124)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2918..3506
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3407..3426
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3660..3677
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3692..3834
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3760..3779)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3920..3942
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3936..4127)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4936..6018)
                     /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     5980..5999
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6019..6096)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6302..6321
                     /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 tatggagacc caggctgagc agcaggagct ggaaaccctt ccaaccacca
      481 agatggctca gaccaaccct acgccggggt ccctggggcc atggaagata accatctatg
      541 atcaggagaa ctttcagggc aagaggatgg agttcaccag ctcctgtcca aatgtctctg
      601 agcgcagttt tgataatgtc cggtccctga aggtggaaag tggcgcctgg attggttatg
      661 agcataccag cttctgtggg caacagttta tcctggagag aggagaatac cctcgctggg
      721 atgcctggag tgggagtaat gcctaccaca ttgagcgtct catgtccttc cgccccatct
      781 gttcagctaa tcataaggag tctaagatga ccatctttga gaaggaaaac tttattggac
      841 gccagtggga gatctctgac gactacccct ccttgcaagc catgggctgg ttcaacaacg
      901 aagtcggctc catgaagata caaagtgggg cctgggtttg ctaccaatat cctggatatc
      961 gtgggtatca gtatatcttg gaatgtgacc atcatggagg agactataaa cattggagag
     1021 agtggggctc tcatgcccag acttcgcaga tccaatcgat tcgccgaatc caacagtaat
     1081 ctagaggatc cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac
     1141 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     1201 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1261 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     1321 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1381 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1441 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1501 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1561 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1621 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1681 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1741 taacgtttac aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1801 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1861 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1921 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     1981 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2041 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     2101 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     2161 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     2221 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     2281 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     2341 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     2401 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     2461 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     2521 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     2581 aatctggagc cggtgagcgt ggttctcgcg gtatcattgc agcactgggg ccagatggta
     2641 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     2701 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2761 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2821 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2881 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2941 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3001 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3061 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3121 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3181 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3241 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3301 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3361 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3421 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3481 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3541 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3601 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3661 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     3721 tgtgcggtat ttcacaccgc atatatggtg cactctcagt acaatctgct ctgatgccgc
     3781 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     3841 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3901 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3961 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     4021 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     4081 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     4141 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     4201 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     4261 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     4321 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     4381 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     4441 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     4501 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4561 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     4621 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     4681 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     4741 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     4801 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     4861 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     4921 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4981 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     5041 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     5101 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     5161 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     5221 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     5281 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     5341 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     5401 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     5461 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     5521 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     5581 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     5641 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     5701 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     5761 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     5821 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     5881 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     5941 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     6001 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     6061 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     6121 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     6181 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     6241 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     6301 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     6361 cgatgcgtcc ggcgtagagg atcg
//
