LOCUS       sequence-464161-        6002 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of ubiquitin-conjugating enzyme E2 
            variant 1 (UEV-1) (CROC-1) in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6002)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6002)
  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"
COMMENT     Sequence Label: pAIP4_2A4D
FEATURES             Location/Qualifiers
     source          1..6002
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(36..851)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(36..851)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     759..778
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     843..862
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      973..1561
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1462..1481
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1715..1732
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1747..1886
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1812..1831)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1972..1994
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1988..2179)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2988..4070)
                     /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     4032..4051
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4071..4148)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4354..4373
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4461..4480
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4461..4479
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4480..4504
                     /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             4519..4541
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4564..4593
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5323..5340
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5393..5411)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5407..5454
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5491..5946
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5578..5597)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5788..5809
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
       61 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
      121 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
      181 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
      241 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
      301 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
      361 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
      421 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
      481 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
      541 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
      601 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
      661 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
      721 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
      781 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
      841 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
      901 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
      961 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     1021 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     1081 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     1141 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     1201 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     1261 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     1321 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     1381 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     1441 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     1501 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     1561 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     1621 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     1681 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     1741 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     1801 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     1861 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     1921 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     1981 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     2041 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     2101 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     2161 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     2221 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     2281 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     2341 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     2401 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     2461 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     2521 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     2581 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     2641 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     2701 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     2761 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     2821 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     2881 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     2941 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     3001 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3061 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     3121 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     3181 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     3241 tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta
     3301 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     3361 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     3421 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     3481 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     3541 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     3601 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     3661 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     3721 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     3781 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     3841 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     3901 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     3961 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     4021 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     4081 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     4141 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     4201 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     4261 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     4321 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     4381 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     4441 cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     4501 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat gatggcgaag
     4561 attcatcacc atcatcacca tcaccatcac cacggtagcc tgcaagattc ggaagtcaac
     4621 caagaagcaa agccggaagt caagccggaa gtgaaaccgg aaacccatat taacctgaaa
     4681 gttagtgacg gcagctctga aattttcttt aagatcaaaa agaccacgcc gctgcgtcgc
     4741 ctgatggaag cgtttgccaa acgtcagggc aaggaaatgg atagcctgcg tttcctgtat
     4801 gacggtattc gcatccaggc agatcaagcg ccggaagacc tggacatgga agacaacgac
     4861 atcattgaag cccaccgtga acagattggc ggctctggag tacccagaaa ttttaggcta
     4921 ttagaagagt tggaggaagg ccagaaaggt gtgggtgatg gtactgtaag ctggggtctg
     4981 gaagacgacg aagacatgac cctgacccgt tggacgggta tgattatcgg ccctccgcgc
     5041 accatttatg agaaccgtat ctactccctg aagatcgagt gcggtccgaa gtacccggag
     5101 gctccgccat ttgttcgttt cgtgaccaaa atcaacatga acggcgttaa ttcgagcaat
     5161 ggcgtggtgg atccgcgcgc aattagcgtt ttggcgaagt ggcagaattc ttacagcatt
     5221 aaagttgtcc tccaagaact gagacgtctg atgatgtcca aagaaaacat gaagctgccg
     5281 cagccgccgg agggccaatg ttatggttcg taatgactcg agcaccacca ccaccaccac
     5341 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     5401 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     5461 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     5521 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     5581 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5641 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5701 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5761 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5821 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     5881 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     5941 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     6001 aa
//
