DNA molecules are a promising data storage medium for the future; however, effective de novo synthesis of DNA using an enzyme that catalyzes the polymerization of natural nucleoside triphosphates in a user-defined manner, without the need for multiple injections of polymerase, remains a challenge. In the present study, we demonstrated that the bacteriophage abortive infection system reverse transcriptase AbiK from Lactococcus lactis facilitates such an approach. We employed surface plasmon resonance to monitor the polymerization of the DNA strand with a user-defined sequence of multiple segments through a sequential buffer exchange process. Using this method, we synthesized synthetic DNA with segments of random length and a sequence consisting of only three of the four natural nucleotides. The information is encoded using the absence of one nucleotide in each segment. We demonstrated that synthetic DNA can be stored on the chip, and when the DNA is released from the chip, the second strand can be synthesized and read by sequencing. Our setup facilitates a writing speed of one nucleotide in less than 1 s and holds enormous potential for synthesizing DNA for data storage.