LOCUS       sequence-152173-        6383 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 6383)
  TITLE     Larry David lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6383)
  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            "Unpublished"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..6383
                     /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             1131..1148
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1201..1219)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1215..1262
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1299..1754
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1386..1405)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1596..1617
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1781..1885
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1886..1954,1955..2746)
                     /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(2104..2123)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2917..3505
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3406..3425
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3659..3676
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3691..3833
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3759..3778)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3919..3941
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3935..4126)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4935..6017)
                     /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     5979..5998
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6018..6095)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6301..6320
                     /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 caacagtaac
     1081 tagaggatcc gaattcgagc tccgtcgaca agcttgcggc cgcactcgag caccaccacc
     1141 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     1201 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     1261 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     1321 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     1381 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     1441 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     1501 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     1561 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     1621 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     1681 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     1741 aacgtttaca atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1801 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1861 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     1921 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     1981 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     2041 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     2101 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     2161 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2221 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2281 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     2341 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     2401 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     2461 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2521 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2581 atctggagcc ggtgagcgtg gttctcgcgg tatcattgca gcactggggc cagatggtaa
     2641 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2701 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2761 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2821 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2881 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2941 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3001 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3061 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3121 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3181 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3241 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3301 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3361 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3421 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3481 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3541 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3601 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3661 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3721 gtgcggtatt tcacaccgca tatatggtgc actctcagta caatctgctc tgatgccgca
     3781 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3841 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3901 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3961 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     4021 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     4081 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4141 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4201 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4261 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     4321 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     4381 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4441 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     4501 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4561 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     4621 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     4681 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     4741 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     4801 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     4861 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     4921 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4981 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     5041 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     5101 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     5161 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     5221 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     5281 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     5341 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     5401 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     5461 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     5521 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     5581 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     5641 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     5701 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     5761 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     5821 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     5881 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     5941 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     6001 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     6061 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     6121 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     6181 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     6241 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     6301 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     6361 gatgcgtccg gcgtagagga tcg
//
