LOCUS       sequence-153530-        6327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human beta A2-crystallin in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6327)
  TITLE     Larry David lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6327)
  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            "Unpublished"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 CRYBA2
FEATURES             Location/Qualifiers
     source          1..6327
                     /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             1075..1092
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1145..1163)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1159..1206
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1243..1698
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1330..1349)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1540..1561
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1725..1829
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1830..1898,1899..2690)
                     /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(2048..2067)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2861..3449
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3350..3369
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3603..3620
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3635..3777
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3703..3722)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3863..3885
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3879..4070)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4879..5961)
                     /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     5923..5942
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5962..6039)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6245..6264
                     /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 tagcagcgcc cccgcgccgg gcccggcgcc cgccagcctc acgctctggg
      481 acgaggagga cttccagggc cgtcgctgtc ggctgctaag cgactgtgcg aacgtctgcg
      541 agcgcggagg cctgcccagg gtgcgctcgg tcaaggtgga aaacggcgtt tgggtggcct
      601 ttgagtaccc cgacttccag ggacagcagt tcattctgga gaagggagac tatcctcgct
      661 ggagcgcctg gagtggcagc agcagccaca acagcaacca gctgctgtcc ttccggccag
      721 tgctctgcgc gaaccacaat gacagccgtg tgacactgtt tgagggggac aacttccaag
      781 gctgcaagtt tgacctcgtt gatgactacc catccctgcc ctccatgggc tgggccagca
      841 aggatgtggg ttccctcaaa gtcagctccg gagcgtgggt ggcctaccag tacccaggct
      901 accgaggcta ccagtatgtg ttggagcggg accggcacag cggagagttc tgtacttacg
      961 gtgagctcgg cacacaggcc cacactgggc agctgcagtc catccggaga gtccagcact
     1021 aatctagagg atccgaattc gagctccgtc gacaagcttg cggccgcact cgagcaccac
     1081 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     1141 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1201 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     1261 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1321 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1381 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1441 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1501 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1561 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1621 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1681 tattaacgtt tacaatttca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1741 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1801 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1861 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1921 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1981 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     2041 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     2101 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     2161 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     2221 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     2281 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     2341 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     2401 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2461 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     2521 ataaatctgg agccggtgag cgtggttctc gcggtatcat tgcagcactg gggccagatg
     2581 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2641 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2701 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2761 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2821 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2881 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2941 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3001 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3061 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3121 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3181 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3241 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3301 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3361 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3421 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3481 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3541 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3601 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3661 atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc
     3721 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3781 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3841 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3901 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     3961 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     4021 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     4081 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     4141 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     4201 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     4261 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     4321 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     4381 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     4441 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4501 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     4561 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     4621 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     4681 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     4741 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     4801 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4861 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4921 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4981 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     5041 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     5101 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     5161 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     5221 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     5281 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     5341 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     5401 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     5461 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5521 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5581 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5641 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5701 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5761 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5821 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5881 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5941 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     6001 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     6061 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     6121 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     6181 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     6241 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     6301 ccacgatgcg tccggcgtag aggatcg
//
