LOCUS       sequence-434867-        6488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of SUMO tagged human beta B1 crystallin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6488)
  TITLE     Completion of previous SUMO tagged human crystallin submission plus 
            plasmids for native chemical ligation to produced gamma S crystallin
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6488)
  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            "Unpublished"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 CRYBB1
FEATURES             Location/Qualifiers
     source          1..6488
                     /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             596..607
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             1240..1257
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1310..1328)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1324..1371
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1408..1863
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1495..1514)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1705..1726
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1889..1993
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1994..2062,2063..2854)
                     /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(2212..2231)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3025..3613
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3514..3533
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3767..3784
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3799..3938
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3864..3883)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4024..4046
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4040..4231)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5040..6122)
                     /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     6084..6103
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6123..6200)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6406..6425
                     /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 ttctcaggct gcaaaggcct cggcctcggc cacagtggcg gtgaacccag
      481 ggcctgacac caaggggaag ggggccccac ctgcaggaac atcccctagt cccggcacta
      541 ccctggcccc aacaaccgtg cctattacca gcgccaaggc ggcggaactg cctcctggga
      601 actacaggct ggtggtcttc gaactggaaa acttccaggg ccgtcgagca gaattctcgg
      661 gggagtgctc aaatctggca gaccgtggct tcgaccgtgt gcgcagcatc attgtctccg
      721 cgggaccctg ggtcgccttt gagcagtcca acttccgcgg ggagatgttc atcctggaga
      781 agggcgagta ccctcgctgg aacacatggt cgagcagcta ccgcagtgat cggctcatgt
      841 ccttccggcc catcaaaatg gatgcccagg agcacaaaat ctccctgttt gaaggggcca
      901 acttcaaggg caacaccata gagatccagg gggacgacgc acccagtctc tgggtctacg
      961 gcttcagtga ccgcgtgggc agcgtgaagg tctccagtgg aacatgggtt ggctatcagt
     1021 atcctggcta ccgcgggtac cagtacctcc tagagcctgg tgacttccgg cactggaatg
     1081 agtggggagc cttccagcca cagatgcagt ccctgcgtcg cctgcgtgac aagcagtggc
     1141 acctcgaggg gtccttccct gtcctggcca cagagccccc caagtaatct agaggatccg
     1201 aattcgagct ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga
     1261 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     1321 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     1381 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     1441 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1501 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1561 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1621 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1681 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1741 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1801 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     1861 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1921 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1981 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     2041 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     2101 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     2161 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     2221 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     2281 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     2341 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     2401 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     2461 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     2521 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     2581 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     2641 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     2701 tgagcgtggt tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     2761 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     2821 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     2881 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     2941 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3001 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3061 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3121 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3181 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3241 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3301 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3361 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3421 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3481 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3541 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3601 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3661 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3721 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3781 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3841 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     3901 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     3961 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4021 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4081 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     4141 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     4201 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     4261 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     4321 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     4381 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     4441 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     4501 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     4561 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     4621 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     4681 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     4741 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     4801 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     4861 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     4921 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     4981 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     5041 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5101 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     5161 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     5221 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     5281 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     5341 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     5401 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     5461 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     5521 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     5581 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     5641 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     5701 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     5761 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     5821 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     5881 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     5941 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     6001 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     6061 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     6121 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     6181 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     6241 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     6301 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     6361 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     6421 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     6481 gaggatcg
//
