LOCUS       sequence-434869-        6260 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of SUMO tagged human alpha B crystallin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6260)
  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  JOURNAL   Unpublished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 6260)
  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            "Unpublished"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 CRYAB
FEATURES             Location/Qualifiers
     source          1..6260
                     /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             1012..1029
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1082..1100)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1096..1143
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1180..1635
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1267..1286)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1477..1498
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1661..1765
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1766..1834,1835..2626)
                     /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(1984..2003)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2797..3385
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3286..3305
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3539..3556
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3571..3710
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3636..3655)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3796..3818
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3812..4003)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4812..5894)
                     /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     5856..5875
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5895..5972)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6178..6197
                     /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 tatggacatc gccatccacc acccctggat ccgccgcccc ttctttcctt
      481 tccactcccc cagccgcctc tttgaccagt tcttcggaga gcacctgttg gagtctgatc
      541 ttttcccgac gtctacttcc ctgagtccct tctaccttcg gccaccctcc ttcctgcggg
      601 cacccagctg gtttgacact ggactctcag agatgcgcct ggaaaaggac aggttctctg
      661 tcaacctgga tgtgaagcac ttctccccag aggaactcaa agttaaggtg ttgggagatg
      721 tgattgaggt gcatggaaaa catgaagagc gccaggatga acatggtttc atctccaggg
      781 agttccacag gaaataccgg atcccagctg atgtagaccc tctcaccatt acttcatccc
      841 tgtcatctga tggggtcctc actgtgaatg gaccaaggaa acaggtctct ggccctgagc
      901 gcaccattcc catcacccgt gaagagaagc ctgctgtcac cgcagccccc aagaaataat
      961 ctagaggatc cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac
     1021 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     1081 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1141 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     1201 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1261 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1321 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1381 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1441 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1501 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1561 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1621 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1681 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1741 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     1801 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     1861 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     1921 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     1981 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     2041 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2101 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2161 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     2221 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     2281 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     2341 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2401 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2461 atctggagcc ggtgagcgtg gttctcgcgg tatcattgca gcactggggc cagatggtaa
     2521 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2581 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2641 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2701 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2761 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2821 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2881 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2941 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3001 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3061 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3121 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3181 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3241 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3301 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3361 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3421 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3481 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3541 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3601 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3661 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     3721 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3781 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3841 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     3901 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     3961 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     4021 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     4081 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     4141 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     4201 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     4261 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     4321 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     4381 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     4441 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     4501 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     4561 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     4621 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     4681 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     4741 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     4801 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4861 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     4921 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     4981 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     5041 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     5101 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     5161 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     5221 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     5281 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     5341 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     5401 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     5461 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     5521 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     5581 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     5641 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     5701 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     5761 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     5821 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     5881 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     5941 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     6001 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     6061 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     6121 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     6181 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     6241 gcgtccggcg tagaggatcg
//
